Tesis-bases Moleculares De La Especificidad Peptídica De Hla-b27

Universidad Autónoma de Madrid Facultad de ciencias Departamento de Biología Molecular

BASE MOLECULAR DE LA ESPECIFICIDAD PEPTÍDICA DE HLA-B27

JOSÉ RAMÓN LAMAS LÓPEZ Madrid, 1999

Universidad Autónoma de Madrid Facultad de ciencias Departamento de Biología Molecular

Base molecular de la especificidad peptídica de HLA-B27

Memoria para optar al grado de Doctor en Ciencias presentada por: José Ramón Lamas López Director: Dr. José Antonio López de Castro Álvarez Profesor de investigación del C.S.I.C Centro de Biología Molecular "Severo Ochoa"

Madrid, Junio de 1999.

ABREVIATURAS

APC

Células presentadoras de antígeno.

AR

Artritis reactiva

Aua

Ácido 11-amino undecanoico.

EA

Espondilitis anquilosante.

EBNA

Antígeno nuclear del EBV.

EBV

Virus de Epstein-Barr.

β 2m

β2-microglobulina.

BCR

Receptor de células B.

CD

Cluster of Differentiation.

C-terminal

Extremo Carboxilo.

CTL

Linfocito T citotóxico.

HB

(R)-3-Hidroxibutirato.

HLA

Antígenos Leucocitarios Humanos.

Kb

Kilobase.

kDa

Kilodalton.

LMP

Proteína latente de membrana del EBV

MHC

Complejo principal de histocompatibilidad.

N-terminal

Extremo amino.

PX

Posición X del péptido.

PΩ

Posición C-terminal.

RE

Retículo Endoplásmico.

TAP

Transportador asociado con la presentación de antígeno.

TCR

Receptor de célula T.

GLICINA

ASPARAGINA

GLUTAMINA

METIONINA

CISTEÍNA

(Gly)

(Asn)

(Gln)

(Cys)

G

N

Q

(Met) M

COO+H N C H 3

COO+H N C H 3

H

COO+H N C H 3

COO+H N C H 3

C COO+H N C H 3

CH2

CH2

CH2

CH2

C

CH2

CH2

SH

C

S

NH2

O

NH2

O

CH3

TREONINA

SERINA

TIROSINA

FENILALANINA

TRIPTÓFANO

(Thr)

(Ser)

(Tyr)

(Phe)

(Trp)

T

S

Y

F

W

COO+H N C H 3 H C OH

COO+H N C H 3

COO+H N C H 3

H C OH

CH3

COO+H N C H 3

CH2

COO+H N C H 3

CH2

CH2

H NH OH Aminoácidos polares (sin carga)

Aminoácidos apolares aromáticos

ALANINA

VALINA

LEUCINA

ISOLEUCINA

PROLINA

(Ala)

(Val)

(Leu)

(Ile)

(Pro)

A

V

L

I

P

COO+H N C H 3

COO+H N C H 3

CH3

COO+H N C H 3

CH H3C

CH3 H3C

COO+H N C H 3

CH2

H C CH3

CH

CH2

CH3

CH3

COO+H N C H 2 H2C

CH2 CH2

Aminoácidos apolares alifáticos Ác. ASPÁRTICO

Ác. GLUTÁMICO

LISINA

HISTIDINA

ARGININA

(Asp)

(Glu)

(Lys)

(His)

(Arg)

D

E

K

H

R

COO+H N C H 3

COO+H N C H 3

CH2 C O

O

-

CH2

CH2

CH2

CH2

C O

COO+H N C H 3

CH2 O

-

COO+H N C H 3 CH2 +HN

NH

CH2 NH 3+

Aminoácidos polares (con carga)

COO+H N C H 3 CH2 CH2 CH2 NH C=NH 2+ NH2

ÍNDICE

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I. INTRODUCCIÓN. ...................................................................................................................................................................................... 1 I.1. Características generales del sistema inmunitario.

.................................................................................................... 3

I.2. Organización genómica y estructural del MHC humano.

...................................................................................... 4

I.2.1. Organización de los genes del MHC de clase I y clase II. I.3. Estructura de las proteínas de clase I.

...................................................................... 4

............................................................................................................................. 6

I.3.1. La cadena pesada y la Beta-2 microglobulina (β2m). . .............................................................................. 6 I.3.2. Los péptidos. ................................................................................................................................................................ 8 I.4. Procesamiento de antígeno. Formación, transporte y presentación de los complejos péptido-MHC de clase I. .......................................................................................................................................................... 9 I.4.1. Origen y procesamiento de los péptidos antigénicos. ................................................................................ 9 I.4.2. Translocación al Reticulo Endoplásmico.

...................................................................................................... 10

I.4.3. Biosíntesis, Ensamblaje y expresión en membrana de las moléculas de clase I.

........................... 11

I.5. TCR y reconocimiento de los complejos MHC-péptido. ........................................................................................... 11 I.5.1. Características estructurales del TCR y reconocimiento de los complejos MHC-péptido. I.6. HLA-B27.

....... 11

...................................................................................................................................................................................... 13

I.6.1. Subcavidad A. ............................................................................................................................................................. 13 I.6.2. Subcavidad B. ............................................................................................................................................................. 13 I.6.3. Subcavidades C y F.

................................................................................................................................................ 14

I.6.4. Subcavidades D y E.

................................................................................................................................................ 15

I.7. HLA-B27 y espondiloartropatías. ....................................................................................................................................... 15 I.7.1. Posible papel patogénico de la presentación de péptidos por HLA-B27.

......................................... 16

I.7.2. Distribución étnica y asociación a enfermedad de los subtipos de HLA-B27. II. OBJETIVOS.

................................ 17

............................................................................................................................................................................................... 21

III. MATERIALES Y MÉTODOS.

.......................................................................................................................................................... 25

III.1. Líneas celulares. ..................................................................................................................................................................................27 III.2. Anticuerpos monoclonales (mAb).

............................................................................................................................................27

III.3. Síntesis, purificación y cuantificación de péptidos. ..........................................................................................................27 II.3.1. Síntesis y purificación de péptidos y análogos no peptídicos ........................................................................27 III.3.2. Cuantificación. ...................................................................................................................................................................28 III.4. Ensayo de unión de péptidos.

.......................................................................................................................................................28

III.4.1. Análisis por citometría de flujo.

................................................................................................................................29

III.4.2. Cálculo de la unión de péptidos. ...............................................................................................................................29 IV. RESULTADOS.

........................................................................................................................................................................................ 31

IV.1 Efecto del polimorfismo de HLA-B27 sobre la especificidad de unión de péptidos. . .......................................33 IV.1.1. Unión de péptidos a HLA-B*2705, B*2704 y B*2706. Modulación de la especificidad por el residuo peptídico C-terminal. ........................................................................................................................33

IV.1.1.1. Efectos de la pérdidas y ganancias de residuos cargados en las subcavidades E/C/F. ..................................................................................................................................34

ÍNDICE

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IV.1.2. Unión de péptidos a B*2701 y B*2702. Efecto del polimorfismo sobre la especicificidad de los residuos en P2. ........................................................................................................ 37 IV.1.3. Unión de péptidos a B*2703:Papel del polimorfismo de la subcavidad A. ........................................ 40 IV.1.3.1. Propiedades dinámicas vs afinidad de unión de péptidos.

................................................. 40

IV.1.3.2. Fluctuaciones atómicas, áreas accesibles y no accesibles.

............................................... 41

IV.1.3.3. Analisis cualitativo y cuantitativo de los puentes de hidrógeno.

..................................... 42

IV.2. Relación entre la unión de péptidos y la selección de epítopos virales por células T.

............................. 43

IV.2.1. Unión de péptidos virales a diferentes subtipos deHLA-B27, y su relación con la inmunogenicidad. ....................................................................................................................... 43 IV.2.2. Reconocimiento de péptidos de EBV por CTLs restringidos por HLA-B27.

.................. 44

IV.2.3. El motivo Arg2, anclaje principal de los péptidos unidos a HLA-B27, no es esencial para mantener la capacidad antigénica del péptido. .............................................. 44 IV.3. Modulación de la especificidad en las posiciones de anclaje P1, P3 y PΩ, por el polimorfismo de HLA-B27. ............................................................................................................................................. 47 IV.3.1. Especificidad de B*2705, B*2704 y B*2706 por los residuos en P1, P3 y P9. .............. 47 IV.3.2. La unión de un péptido es el resultado de la contribución aditiva de varios residuos de anclaje. .................................................................................................................................. 48 IV.3.3. Distribución de los residuos P1, P3 y PΩ. entre los ligandos naturales de B*2705. . 52 IV.4. Unión de análogos no peptídicos a HLA-B27.

.......................................................................................................... 55

IV.4.1. Reemplazamiento de la parte central de epítopos naturales con un espaciador monofuncional. .............................................................................................................. 55 IV.4.2. Reemplazamiento de la parte central de epítopos naturales con espaciadores bifuncionales. ............................................................................................................. 56 IV.4.3. Modelado molecular de la unión de análogos con espaciadores no peptídicos a B*2705. ........................................................................................................................... 56 V. DISCUSIÓN. ................................................................................................................................................................................................. 59 V.1. Polimorfismo de HLA-B27 y solapamiento de repertorios peptídicos entre subtipos.

.............................. 61

V.1.1. Influencia del polimorfismo de la cavidad C/F sobre la especificidad por el extremo C-terminal del péptido. Diferencias entre B*2704 y B*2706. ............................. 61 V.1.2. El polimorfismo de la cavidad C/F determina parcialmente la especificidad de la subcavidad B. . ................................................................................................................................ 63 V.1.3. Análisis del efecto del polimorfismo de la subcavidad A sobre la unión de péptidos. ........................................................................................................................................................ 65 V.2. Relación entre la unión de péptidos y la selección de epítopos por células T. .............................................. 67 V.3. La especificidad de péptidos por los subtipos de HLA-B27 es modulada en múltiples posiciones de anclaje. ............................................................................................................................................................ 70 V.4. Unión de análogos no peptídicos a HLA-B27. Resumen y discusión general.

............................................................................................................ 72

...................................................................................................................................................... 73

VI. CONCLUSIONES.

.................................................................................................................................................................................. 75

VII. REFERENCIAS.

.................................................................................................................................................................................... 79

VIII. ANEXOS.

................................................................................................................................................................................................. 91

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I. INTRODUCCIÓN

INTRODUCCIÓN

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I. INTRODUCCIÓN I.1. CARACTERÍSTICAS GENERALES DEL SISTEMA INMUNITARIO.

E

l sistema inmunitario es el encargado de proteger a un organismo discriminando entre lo que le pertenece y lo extraño a él. Cuando esta función falla y lo propio no es reconocido como tal, se desencadena una respuesta autoinmune.

Funcionalmente, se distinguen dos mecanismos inmunitarios perfectamente coordinados.

El sistema inmune innato o no adaptativo, genera respuestas rápidas e inespecíficas dirigidas hacia características generales del agente patógeno, creando una primera barrera defensiva tanto a nivel físico y bioquímico, a cargo de la piel, mucosas, secreciones y diferentes factores solubles, como celular por células fagocíticas (polimorfonucleares neutrófilos, monocitos y macrófagos), células citotóxicas NK (Natural Killer) o células secretoras (eosinófilos, basófilos y mastocitos). El sistema inmune específico o adaptativo a diferencia del anterior, está adaptado a la naturaleza del patógeno, a su estrategia invasora y posee memoria, lo que le permite generar una respuesta más potente y rápida ante una reexposición al antígeno que provocó la respuesta inicial. Los mecanismos efectores de la respuesta adaptativa dependen de receptores expresados en los linfocitos B y T que reconocen antígenos de diferentes características. Los receptores de los linfocitos B o BCR (B Cell Receptor) reconocen el antígeno en su estado nativo, activando la diferenciación y maduración de los linfocitos B para secretar las inmunoglobulinas (Ig) o anticuerpos, una versión soluble del receptor que mantiene su especificidad original. Los receptores de los linfocitos T o TCR (T Cell Receptor), reconocen fragmentos peptídicos derivados del procesamiento del antígeno, unidos a moléculas del MHC (Major Histocompatibility Complex). Para ello requieren la presencia de células presentadoras encargadas de procesar el antígeno y de presentarlo en la superficie celular para su reconocimiento por los receptores antigénicos de los linfocitos T. Basándose en su estructura y función se distinguen dos tipos de moléculas del MHC: de clase I y de clase II.

INTRODUCCIÓN

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Los linfocitos T se distribuyen en dos subpoblaciones dependiendo del tipo de proteína correceptora, CD4 o CD8, que expresan en su membrana. Estos correceptores en general no se expresan juntos en un mismo linfocito T diferenciado, y la presencia de uno u otro determina diferentes patrones de restricción por MHC (Zinkernagel y Doherty, 1974). Los linfocitos T citotóxicos (CD8+) o CTLs (Citotoxic T Lymphocytes), son células efectoras líticas, que reconocen el péptido presentado por moléculas del MHC de clase I, se dice que están restringidas por el MHC de clase I. Las células Th (helper) o reguladoras (CD4+), están restringidas por el MHC de clase II. El reconocimiento de péptidos unidos a estas moléculas inicia una serie de procesos esenciales para desencadenar una respuesta inmune, puesto que de ello depende tanto la diferenciación de los linfocitos B en células plasmáticas secretoras de anticuerpos, como la activación y diferenciación de los linfocitos T CD8+ en células efectoras. I.2. ORGANIZACIÓN GENÓMICA Y ESTRUCTURAL DEL MHC HUMANO. Las moléculas del MHC, denominadas en humanos HLA (Human Leukocyte Antigens), son codificadas por un conjunto de genes agrupados en una zona de aproximadamente 3.500 Kb, en el brazo corto del cromosoma 6 (Francke y Pellegrino, 1977; Dunham et al., 1987) donde se diferencian tres regiones: de clase I, II y III. I.2.1. Organización de los genes del MHC de clase I y clase II. La región de clase I abarca una extensión de 1600 Kb orientada hacia el extremo telomérico, donde se emplazan los loci codificantes de las cadenas α o cadenas pesadas de los antígenos de clase Ia o clásicos (HLA-A, -B y -C) y los de clase Ib o no clásicos (HLA-E, F, G) (Figura 1). Los genes que codifican las cadenas pesadas de los antígenos de clase Ia, son muy polimórficos (Parham et al., 1995) y se expresan en la mayoría de las células somáticas de forma codominante y a niveles de expresión variables, aunque típicamente elevados en las células de linaje hematopoyético. Por el contrario, los genes codificantes de las moléculas de clase Ib, son poco polimórficos y su expresión tisular está más limitada (Wei y Orr, 1990). Estructuralmente son semejantes a los antígenos de clase I clásicos pero reconocen antígenos de diferente naturaleza. Entre estos antígenos de clase Ib, se incluyen además, las moléculas CD1 (Melián et al., 1996) aunque son codificadas por el cromosoma 1, fuera del MHC. Recientemente se ha identificado una familia de cinco genes ubicados en las regiones de clase I y III denominada

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INTRODUCCIÓN

MIC (MHC class I Chain related) (Bahram et al., 1994), que codifican cadenas pesadas con secuencias muy divergentes de las otras cadenas de clase I codificadas en el MHC. Orientada hacia el extremo centromérico, la región de clase II tiene una amplitud de 900 Kb, donde se ubican los loci HLA-DP, -DQ y -DR que codifican a las cadenas α y β de las moléculas de clase II. Estas moléculas se expresan exclusivamente en macrófagos, células dendríticas y células B activadas, conocidas como células presentadoras de antígeno “profesionales” o APCs (Antigen-Presenting Cells). En esta región se localizan además otros genes que codifican a varias proteínas implicadas en el procesamiento, transporte y presentación de antígeno como TAP1 y TAP2 (Spies et al., 1990; Trowsdale et al., 1990) la Tapasina (Herberg et al., 1998) y las subunidades del proteosoma LMP2 y LMP7 (Large Multifunctional Proteasome). (Kelly et al., 1991; Glynne et al., 1991). Entre las regiones de clase I y clase II, se sitúa la región de clase III. Esta región de unas 1000 Kb, comprende a un grupo de genes denominados en conjunto genes de clase III, que codifican entre otras, a las proteínas de los factores del complemento C2, C4, B y F y los factores de necrosis tumoral TNFα y TNFβ (Tumor Necrosis Factor α y β).

6

Clase II

β α

DQ

Tapasina

β α

DM

LMP TAP

DP

Clase III

β α

DR

β β α

Factores del complemento

Clase I

BC

E

A GF

TNFα TNFβ

MIC B MIC A

MIC C

MIC D MIC E

Figura 1: Mapa genético del complejo principal de histocompatibilidad humano, ubicado en el brazo corto del cromosoma 6.

INTRODUCCIÓN

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I.3. ESTRUCTURA DE LAS PROTEÍNAS DE CLASE I. Las moléculas HLA de clase I, son glicoproteínas de membrana formadas por la asociación no covalente de tres componentes: una cadena pesada, HC (Heavy Chain) o cadena α, de 45 kDa, codificada en el MHC, una cadena ligera de 12 kDa denominada Beta-2 microglobulina (β2m) (Ploegh et al., 1981), codificada en el cromosoma 15 (Goodfellow et al., 1975) y un péptido antigénico de entre ocho y once aminoácidos. Sus funciónes inmunológicas son la de actuar como receptoras y presentadoras de péptidos al TCR y la de ser reconocidas por receptores de células NK (Ljunggren y Kärre, 1990a; Moretta et al., 1994; Phillips et al., 1996) I.3.1. La cadena pesada y la Beta-2 microglobulina (β2m). Los genes que codifican la cadena pesada de las moléculas de clase I se distribuyen en ocho exones de diferente longitud separados por siete intrones (Jordan et al., 1985). Cada exón codifica fundamentalmente un dominio de la proteína. El exón 1, situado en el extremo 5’ del gen, codifica una porción no traducida de 18 pares de nucleótidos y un péptido señal de 24 aminoácidos. Los exones 2 y 3 (270 pares de nucleótidos) y 4 (276 pares de nucleótidos) codifican los dominios extracelulares de la cadena pesada, α1, α2, y α3 respectivamente. El exón 5 (117 pares de nucleótidos) codifica la región transmembrana y los residuos que la flanquean. Los exones 6, 7 y 8 (33, 48 y 400 pares de nucleótidos respectivamente) codifican la región intracitoplásmica y la región 3’ no traducida. La cadena pesada resultante, consta de aproximadamente 340 aminoácidos estructurados en tres regiones bien definidas: una región amino terminal extracelular de 274 aminoácidos, formada por los dominios α1, α2 y α3 de aproximadamente 90 aminoácidos cada uno, una región transmembrana hidrofóbica de 25 aminoácidos y una intracitoplásmica hidrofílica de unos 30 aminoácidos (Ploegh et al., 1981). Mediante técnicas de difracción de rayos X, ha podido determinarse la similar organización tridimensional de estos dominios en distintas moléculas de clase I, humanas y murinas (Bjorkman et al., 1987; Garrett et al., 1989; Saper et al., 1991; Madden et al., 1991; Madden et al., 1992; Fremont et al., 1992; Zhang et al., 1992; Young et al., 1994; Smith et al., 1996b; Smith et al., 1996c). Los dominios α1 y α2, que son estructuralmente idénticos, constan cada uno de una lámina β antiparalela formada por cuatro cadenas polipeptídicas y de una larga región en α-hélice (Figura 2). Tras el plegamiento de ambos dominios las dos estructuras en α-

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INTRODUCCIÓN

hélice coronan una base constituida por ocho láminas β-plegadas, delimitando una hendidura, denominada surco de unión del péptido, de aproximadamente 25Å de largo por 10Å de ancho. El polimorfismo de los antígenos de clase I, se concentra principalmente en los residuos situados en el interior y las zonas próximas a este surco, donde se distinguen seis subcavidades (Saper et al., 1991) denominadas A-F. Su forma, tamaño y polaridad, está determinada por los residuos polimórficos que las constituyen, y que generan diferentes especificidades por los péptidos unidos (Figura 2). En el residuo Asn86 del dominio α1 se situa

B

C

A

F D

E

un carbohidrato de 3,3 kDa y un puente disulfuro intradominio en α2 conecta las Cisteínas 101 y 164. El dominio α3, que está muy conservado (Parham et al., 1988), es estructuralmente homólogo a la β2m, y a los dominios constantes de las inmunoglobulinas (Orr et al., 1979). Un puente disulfuro conecta los residuos Cys203 y Cys259 y una corta α-hélice adicional entre los residuos

Figura 2: (Arriba, vista de perfil), diagrama esquemático que representa la estructura tridimensional adoptada por los diferentes dominios de una molécula de clase I. No aparecen representadas ni la región transmembrana ni el fragmento que une los dominios extracelulares a la superficie celular. En la figura inferior (vista apicalmente) se detalla la localización de las diferentes subcavidades distribuidas a ambos lados del surco de unión del péptido.

177-181, conecta este dominio con el α2. Contigua al dominio α3 se encuentra la región transmembrana encargada del anclaje de la molécula a la membrana celular. La β2-microglobulina, de la que sólo se conoce un alelo en humanos, consta de 99 aminoácidos muy conservados entre diferentes especies. Carece de dominio intracitoplásmico y se mantiene asociada a la región extracitoplásmica de la cadena pesada interaccionando con sus tres dominios, lo que determina en gran medida la conformación y estabilidad de ésta (Krangel et al., 1979; Seong et al., 1988).

INTRODUCCIÓN

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La β2m y el dominio α3 se sitúan por debajo de la lámina β de α1/α2 dejando una cavidad entre ellos donde el correceptor CD8 de la célula T interacciona con la molécula de clase I (Salter et al., 1990, Gao et al., 1997). I.3.2. Los péptidos. Los péptidos unidos a las moléculas de clase I, funcionan como una parte integral de las mismas permitiendo su plegamiento y expresión estable en la membrana celular, y participando en la selección de repertorios de células T CD8+ (Abe et al., 1992). La cinética de unión entre un péptido y la molécula de clase I, está gobernada por las constantes de asociación y disociación, a su vez determinantes de su afinidad y estabilidad. Esta última parece ser el factor determinante de la inmunogenicidad de los péptidos (Brooks et al.1998). Desde un punto de vista estructural, la unión de un péptido está determinada por dos características básicas: una es su tamaño, generalmente comprendido entre 8 y 11 aminoácidos (Jardetzky et al., 1991; Falk et al., 1991) y la otra, es la conservación en determinadas posiciones de residuos de anclaje cuya localización y contribución varían en función del polimorfismo de la molécula de clase I a la que se unen. La presencia de un único aminoácido o aminoácidos con cadenas laterales similares en una posición de la secuencia del péptido, define los motivos de anclaje primarios, frecuentemente P2 y el residuo C-terminal (PΩ). Otras posiciones de la secuencia del péptido como P1, P3, y P7, son menos restrictivas respecto al aminoácido que las ocupa, y definen los motivos de anclaje secundarios que contribuyen adicionalmente a la unión del péptido. La unión tiene lugar principalmente entre los extremos N-terminal y C-terminal con los residuos que conforman las subcavidades A y F respectivamente, adoptando una conformación extendida (Madden et al., 1991), y arqueandose en su parte central (Guo et al., 1992), aunque en péptidos más largos puede sobresalir un extremo (Collins et al., 1994). Otras posiciones de la cadena principal del péptido (P2, P3, P7), interaccionan con el resto de las subcavidades (Garrett et al., 1989). En la parte central, que es la más accesible al contacto con el TCR, las cadenas laterales de los residuos P4, P5, P6 y P8 del péptido, pueden adoptar distintas orientaciones en función de su secuencia (Madden et al., 1993).

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INTRODUCCIÓN

I.4. PROCESAMIENTO DE ANTÍGENO. FORMACIÓN, TRANSPORTE Y PRESENTACIÓN DE LOS COMPLEJOS PÉPTIDO-MHC DE CLASE I.

I.4.1. Origen y procesamiento de los péptidos antigénicos. Los linfocitos T, reconocen los antígenos en forma de péptidos unidos a las moléculas del MHC. El origen de la proteína antigénica determina tanto la vía de procesamiento, como el ulterior mecanismo de respuesta inmunitaria. Así, las moléculas del MHC de clase II presentan principalmente péptidos derivados de proteínas de origen exógeno, que son endocitadas por APCs y degradadas en el endosoma, en condiciones de pH ácido. Las moléculas de clase I presentan principalmente péptidos procedentes de la degradación en el citosol de proteínas propias o de secuencias señal (Wei y Cresswell, 1992) por un mecanismo independiente de la vía endocítica o lisosómica (Morrison et al., 1986). La degradación de proteínas citosólicas es un proceso estrechamente regulado para evitar la degradación indiscriminada de proteínas propias. Las formas de “etiquetar” la futura degradación proteica son variadas; unas se basan en características de la propia proteína, como la identidad del aminoácido N-terminal (Townsend et al., 1988; Varshavsky, 1992) o la presencia de secuencias de aminoácidos, como las secuencias "PEST" ricas en Pro, Gln, Ser y Thr (Rogers et al., 1986) o las denominadas “destruction boxes” (Glotzer et al., 1991) que determinan la degradación específica en un determinado momento del ciclo celular. En otras ocasiones, las proteínas que deben ser degradadas, son ubiquitinadas enzimáticamente como paso previo a su degradación específica. La mayor parte de la actividad proteolítica del citosol es realizada por el proteosoma, un complejo proteico multicatalítico con actividad proteasa y dependiente de ATP, encargado del reciclaje de proteínas propias dentro de la célula (Rock et al., 1994, Fentenay et al., 1995). Existen dos variantes funcionales del proteosoma: El proteosoma 20S de 700 kDa, está formado por cuatro anillos superpuestos, de siete subunidades cada uno (Löwe et al., 1995; Groll et al., 1997). Los anillos exteriores poseen subunidades α cuya función es esencialmente estructural y reguladora. Los dos anillos internos poseen subunidades β de función catalítica. Entre los componentes del proteosoma se encuentran las subunidades constitutivas X, Y(δ) y Z. Tras la inducción por interferón-γ (IFN-γ), éstas son sustituidas por las subunidades funcionales LMP7, LMP2 y MECL-1 respectivamente (Kelly et al., 1991; Glynne et al., 1991; Groettrup et al., 1996). Los estudios realizados sobre la función de las subunidades inducibles son poco concluyentes; así, mientras unos sugieren que en la presentación de antígeno no son

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necesarias (Arnold et al., 1992; Momburg et al., 1992; Yewdell et al., 1994), otros indican que en su ausencia la presentación es menor (Cerundolo et al., 1995). En otros estudios estas subunidades parecen alterar el patrón de corte del proteosoma (Gaczynska et al., 1993; Gaczynska et al., 1994; Driscoll et al., 1993), y mientras que LMP7 incrementa la ruptura catalítica tras residuos hidrofóbicos y básicos, LMP2 reduce la capacidad de corte tras residuos ácidos. Por último, en un estudio reciente (Eleuteri et al., 1997) LMP7 favorece alguna de las diferentes actividades catalíticas del proteosoma. En concreto, disminuye la actividad de tipo quimotripsina, de corte tras residuos hidrofobicos, y aumenta la preferencia de corte tras aminoácidos aromáticos o con cadenas laterales ramificadas. La otra variante del proteosoma, encargada de la ruptura de proteínas ubiquitinadas, es la 26S de 1500 kDa. Su núcleo catalítico está formado por el proteosoma 20S asociado a proteínas adicionales que regulan su actividad (Pamer y Cresswell, 1998). Aunque hay evidencias tanto de la existencia de un recorte o “trimming” por endopeptidasas dentro del RE (Retículo Endoplásmico) (Snyder et al., 1994; Hughes et al., 1996), como de la retrotranslocación al citoplasma previamente a su recorte (Roelse et al., 1994), se desconoce la importancia que tiene esta variante generadora de péptidos. I.4.2. Translocación al Retículo Endoplásmico. Una vez generados en el citosol, los péptidos pasan al RE donde se unen a las moléculas de clase I nacientes, para formar complejos péptido/MHC de clase I estables que son translocados hasta la superficie celular. La demostración de que la expresión de las moléculas de clase I era recuperada en células mutantes tras añadir péptido exógeno (Townsend et al., 1989), y de que la expresión era recuperable transfectando dos genes localizados en la subregión de clase II (Spies et al., 1991), sugerían el importante papel del péptido en la estabilización de la molécula de clase I, y la implicación de un transportador responsable de la trasnslocación de péptidos al RE. El responsable resultó ser una proteína heterodimérica, expresada en la membrana del RE, dependiente de ATP y perteneciente a la familia de los ABC (ATP Binding Cassette) (Androlewicz et al., 1994) denominada TAP (Transporter associated with Antigen Processing). El TAP está constituido por la asociación de los monómeros TAP1 y TAP2. Ambos poseen una región N-terminal con múltiples dominios transmembrana, y un dominio C-terminal de unión a ATP.

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INTRODUCCIÓN

TAP muestra preferencia por péptidos de 9-10 aminoácidos, el tamaño canónico de los péptidos unidos a las moléculas de clase I. La identidad del aminoácido C-terminal parece importante en la selectividad del TAP que en humanos une preferentemente péptidos con residuos C-terminales hidrofóbicos y básicos y no acepta Prolina ni Glicina (Momburg et al., 1994). El tamaño de los péptidos translocados por TAP, oscila entre 8 y 13 aminoácidos aunque pueden ser mayores (Androlewicz et al., 1994; Schumacher et al., 1994). La translocación de los péptidos al RE, a diferencia de la unión de péptidos a TAP, es un proceso dependiente de ATP. I.4.3. Biosíntesis, Ensamblaje y expresión en membrana de las moléculas de clase I. Tras la biosíntesis de las proteínas de clase I, en el correcto plegamiento y ensamblaje de HLA intervienen secuencialmente diferentes chaperonas del RE rugoso; éstas actúan como un control de calidad, que impide la asociación de las subunidades plegadas incorrectamente. Las cadenas pesadas de las moléculas de clase I formadas de novo, son retenidas en primera instancia por la Calnexina, una chaperona de membrana de 88 kDa (Jackson et al., 1994; Rajagopalan et al., 1994) que las mantiene parcialmente plegadas hasta que se une la β2m. Una vez formado el heterodímero Cadena α/β2m, se disocia de la Calnexina y se une a otra chaperona similar, aunque no insertada en la membrana, la Calreticulina (Sadasivan et al., 1996) y a la Tapasina (Sadasivan et al., 1996; Ortmann et al., 1997), una glicoproteína transmembranal de 48 kDa. La Tapasina mantiene asociado al heterodímero parcialmente plegado con la subunidad TAP1 en espera de que un péptido translocado se una. La unión del péptido induce un cambio de conformación que estabiliza el complejo trimérico Péptido/Cadena pesada/β2m y desestabiliza la union con TAP, lo que provoca la disociación del complejo. El complejo péptido/MHC de clase I es posteriormente transportado, pasando por el TGN (Trans Golgi Network) y por un sistema de vesículas, hacia la superficie celular. I.5. TCR Y RECONOCIMIENTO DE LOS COMPLEJOS MHC-PÉPTIDO. I.5.1. Características estructurales del TCR y reconocimiento de los complejos MHC-péptido. El receptor de antígeno de los linfocitos T maduros, está compuesto por heterodímeros clonotípicos α:β, el más común en los linfocitos humanos de sangre periférica, o γ:δ. Las subunidades están unidas por puentes disulfuro, formando una estructura muy similar a la del fragmento Fab de una inmunoglobulina. Cada uno de los monómeros se estructura en dos dominios: uno distal muy variable, responsable de la unión a los complejos MHC/Péptido, y otro

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proximal constante. Ambos dominios tienen unido un carbohidrato. Éstos heterodímeros se asocian no-covalentemente al complejo CD3, que está formado por tres polipéptidos: γ, δ y ε; asociados a dos subunidades independientes, las cadenas ζ y η que forman homodímeros ζζ o heterodímeros ζη unidos por puentes disulfuro. CD3 participa en el proceso de transducción de señales al interior celular tras el reconocimiento por el TCR de un complejo MHC/Péptido (Weiss y Littman, 1994) y en el mantenimiento de la expresión del TCR en la superficie celular. Los genes codificantes del TCR se asemejan a los de las inmunoglobulinas, tanto en su secuencia, como en la forma de ensamblarse, mediante reordenamientos de los segmentos génicos que codifican las regiones variables (V), de diversidad (D), de unión (J) (Joining) y constantes (C). La cadena α se origina a partir de reordenamientos de segmentos génicos V-J mientras que la cadena β surge de dos reordenamientos sucesivos V-D-J para generar exones funcionales. Ambos segmentos reordenados se unen durante la maduración del linfocito en el timo, a sus correspondientes regiones C. Tanto en las cadenas TCRα como TCRβ existen cuatro regiones hipervariables, tres de las cuales forman bucles denominados CDRs (ComplementarityDetermining Regions) CDR1, CDR2 y CDR3 que participan directamente en el reconocimiento del complejo MHC/Péptido. Recientemente se ha podido resolver, mediante cristalografía de rayos X, la topología del reconocimiento por el TCR del complejo MHC-péptido (Garboczi et al., 1996; García et al., 1996). Los resultados obtenidos se ajustan en gran medida a predicciones indirectas previas, basadas en el reconocimiento de análogos peptídicos (Jorgensen et al., 1992) y mutantes de moléculas del MHC (Sun et al., 1995). En el modelo cristalográfíco, El TCR cubre la superficie del péptido y parte de la molécula del MHC transversalmente, de manera que el reconocimiento del complejo MHC-péptido es realizado por los CDRs. Cada uno de los CDRs reconoce una parte diferente del complejo. Las regiones CDR3α y CDR3β se sitúan sobre la parte central del péptido y las regiones CDR1α y CDR1β sobre los extremos del péptido N- y C-terminales respectivamente. Las regiones CDR2α y CDR2β interaccionan directamente con los dominios α2 y α1 respectivamente de la molécula del MHC. Este modo de interaccion entre el TCR y los complejos MHC-péptido parece ser general.

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INTRODUCCIÓN

I.6. HLA-B27. El antígeno HLA-B27 es una de las moléculas de clase I humanas mejor estudiadas debido a su fuerte asociación con las espondiloartropatías. El 95% de los pacientes que sufren espondilitis anquilosante (EA), y entre el 60-80% de los que sufren artritis reactiva (AR), son HLA-B27, Hasta el momento se han identificado quince subtipos (Tabla 1) cuyo polimorfismo se localiza fundamentalmente en el sitio de unión del péptido. La estructura cristalográfica del subtipo principal, HLA-B*2705, ha permitido el análisis detallado de las interacciones de esta molécula con péptidos. A continuación se detalla la estructura de las subcavidades del sitio de unión de péptido y la naturaleza de las interacciones entre éstas y las cadenas laterales de los péptidos unidos. I.6.1. Subcavidad A. Esta subcavidad es idéntica en todos los subtipos de HLA-B27 a excepción de B*2703

Subcavidad A

α1

Lámina- β

que presenta el cambio Y59H. El extremo N-terminal del péptido se aloja

Tyr7 Tyr59

P2 P3

P1

en esta subcavidad formando una red pentagonal de puentes de hidrógeno con los residuos conservados Tyr7, Tyr59 y Tyr 171, y a través de una molécula de agua intermedia, con los

Tyr171 Tyr159

α2

residuos Glu63 y Glu45 (Figura 3). El oxígeno del grupo carbonilo de P1 establece un puente de hidrógeno adicional con el residuo Tyr 159. La

Figura 3: Red de puentes de hidrógeno que se establecen en la subcavidad A con el extremo Nterminal del péptido. (Madden et al., 1992).

presencia de His59 en B*2703 provoca una disrupción de esta red induciendo un reforzamiento de las interacciones en la subcavidad B (Villadangos et al., 1995) I.6.2. Subcavidad B. Esta subcavidad está conservada entre los subtipos de HLA-B27 y es diferente en otras moléculas de clase I a excepción de B73 (Parham et al., 1994; Vilches et al., 1994b). Está constituida, entre otros residuos, por His9, Thr24, Glu45, Cys67 y Tyr99 y acomoda la cadena

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lateral del residuo P2 del péptido (Figura 4), que Subcavidad B

en HLA-B27 es Arg y constituye el anclaje

Cys67

peptídico principal (Jardetzky et al., 1991;

Thr24

Glu45

Rammensee et al., 1995).

His9

En el modelo cristalográfico, el grupo guanidinio de la Arg2, establece cinco puentes

P2 Arg

de hidrógeno, tres de ellos directamente con Glu45 y Thr24 y dos indirectos con His9 y Tyr99

Tyr99 PEPTIDO

por mediación de una molécula de agua. El grupo

sulfhidrilo

de

la

Cys67

se

sitúa

exactamente sobre el plano del grupo guanidinio.

Figura 4: Interacciones que tienen lugar entre la cadena lateral de la Arginina y residuos de la subcavidad B. (Madden et al., 1992)

El Glu45 es un residuo crítico en la especificidad de la subcavidad B por Arg2 (Villadangos et. al., 1995). La Lys70, un residuo exclusivo de HLA-B27 presente en casi todos los subtipos, se localiza en las proximidades de esta subcavidad. Sin embargo su cadena lateral se orienta hacia fuera de la subcavidad B porque establece un puente salino con el Asp74. La mutación Tyr74 presente en B*2701 impide la formación de dicho puente salino relajando la conformación de la Lys70, lo que afecta a la especificidad de la subcavidad B y favorece la presencia de Gln en P2 (García et al., 1997c / Anexo 2). I.6.3. Subcavidades C y F. Las subcavidades C y F forman en HLA-B27 una única cavidad denominada C/F, donde el extremo C-terminal del péptido establece una amplia red de puentes de hidrógeno (Figura 5). La mayor parte de las diferencias entre los subtipos de HLA-B27 se concentra en esta cavidad, lo que sugiere una tendencia evolutiva del polimorfismo de HLA-B27 centrado en modular las especificidades por el extremo extremo C-terminal del péptido. En HLA-B27 esta posición constituye junto con la posición 2 otro anclaje primario. En el modelo cristalográfico, el residuo P9, enlaza directamente con Tyr84 y Thr143 e indirectamente, a través de dos moléculas de agua, con Thr80 y Lys 146.

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Las cadenas laterales de los residuos

C-terminales básicos (Guo et al., 1992; Silver et al., 1992). Sin embargo, en HLA-B27, esta situación

no

impide

acomodar

residuos

Subcavidad C/F

α1

Asp77 y Asp116, favorecen la unión de residuos

Asp77

Tyr84

PÉPTIDO P7

P8 P9

hidrofóbicos debido, en parte a la influencia de las cadenas laterales apolares de la Leu81, Tyr123 y Thr143, y al hecho de que los residuos C-terminales

pueden

adoptar

Thr80

Lys146

Thr143 Trp147

α2

distintas

orientaciones y contactar en la subcavidad C/F según su naturaleza química.

Figura 5: Interacciones entre el extremo C-terminal del péptido y los residuos de la cavidad C/F de B*2705. Los residuos polimorficos aparecen subrayados. (Madden et al., 1992).

I.6.4. Subcavidades D y E. Las posiciones P3 y P7 constituyen anclajes secundarios de los péptidos que une HLAB27. La cadena lateral del residuo P3 interacciona en la subcavidad D. Ésta contiene las cadenas laterales aromáticas Tyr99 y Tyr159 y la alifática Leu156 que determinan su naturaleza predominantemente apolar. En B*2705 la subcavidad D, muestra preferencia por residuos con cadenas laterales voluminosas hidrofóbicas. La cadena lateral polar de His114, se sitúa lateralmente a esta subcavidad pudiendo aceptar la cadena lateral de residuos polares. Esta preferencia por el residuo P3 puede variar en los subtipos B*2706, B*2707 y B*2711 donde la posición 114 está ocupada por Asp o Asn. La subcavidad E aloja la cadena lateral del residuo P7. En HLA-A2 (Madden et al., 1993) la cadena lateral de este residuo muestra una gran variabilidad conformacional dependiente de la secuencia completa del péptido que le permite, según el caso, interaccionar con la molécula de clase I o ser accesible al TCR. En la región intermedia del péptido los contactos con la molécula de HLA-B27 son escasos y las cadenas laterales de los residuos P4, P5, P6 y P8 son en general accesibles al contacto con el TCR. I.7. HLA-B27 Y ESPONDILOARTROPATÍAS. Con el término espondiloartropatías seronegativas, se integra a cinco subcategorías clínicas: la espondilitis anquilosante (EA), la artritis reactiva (AR), la artritis psoriásica, la artritis asociada a la enfermedad inflamatoria intestinal y las espondiloartropatías indiferenciadas. Estas

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enfermedades comparten entre otras características, la ausencia de factores reumatoides y una predisposición genética asociada a la presencia de HLA-B27. Sus manifestaciones clínicas se presentan como un conjunto de desordenes reumáticos y extraarticulares que solapan en muchos de sus síntomas. La espondilitis anquilosante (EA), considerada como la manifestación más pura de espondiloartropatía, es una enfermedad crónica que afecta principalmente al esqueleto axial. La expresión de HLA-B27 no es una condición necesaria ni suficiente para desarrollar una espondiloartropatía, pero sí supone un factor de riesgo, sobre todo en el caso de la EA (Brewerton et al., 1973; Schlosstein et al., 1973) y en menor medida en la AR (Brewerton et al., 1974). Evidencias indirectas sugieren además que otros genes pueden incrementar la susceptibilidad a padecer enfermedad (Brown y Wordsworth, 1997). La importancia que juegan los factores ambientales en el desarrollo de la EA, se desconocen; pero en el caso de la AR, está bien documentada la implicación de bacterias intracelulares obligadas o facultativas responsables de las infecciones de los epitelios urogenital (Chlamidia trachomatis), entérico (Yersinia, Sigella, Salmonella o Campylobacter) y respiratorio (Chlamidia pneumoniae). Los estudios realizados en ratas y ratones transgénicos refuerzan la estrecha relación existente entre la expresión de HLA-B27, la flora bacteriana y el desarrollo de artritis. I.7.1. Posible papel patogénico de la presentación de péptidos por HLA-B27. El mecanismo de asociación de HLA-B27 a las espondiloartropatías se desconoce. Entre las diversas hipótesis que intentan explicar el papel patogénico de HLA-B27, tal vez la que está apoyada por evidencias más convincentes es la teoría del péptido artritogénico (Benjamin y Parham, 1990). Esta hipótesis postula que el evento patogénico primario en las espondiloartropatías sería una estimulación antigénica externa (por ejemplo una infección bacteriana) de una respuesta celular citotóxica contra un péptido restringido por HLA-B27. Estos CTLs activados exógenamente reaccionarían cruzadamente con algún péptido propio presentado por HLA-B27, lo que desencadenaría una reacción autoinmune. Las evidencia a favor de esta hipótesis son más o menos circunstanciales. (i) En humanos, ha sido demostrada la existencia de CTLs autorreactivos contra péptidos

del colágeno de tipo II en el líquido sinovial de pacientes con AR, (Gao et al., 1994), y de CTLs específicos de péptidos bacterianos restringidos por HLA-B27 en pacientes con AR y EA (Hermann et al., 1993, Duchman et al., 1996. Ugrinovic et al., 1997).

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(ii) En ratas transgénicas, la expresión de un elevado número de copias de HLA-B27, es suficiente para el desarrollo de una enfermedad inflamatoria espontánea, con muchas características similares a las espondiloartropatías humanas. El papel de las bacterias en este modelo es evidente puesto que la enfermedad no se desarrolla en condiciones libres de gérmenes (Hammer et al., 1990; Taurog et al., 1994; Breban et al., 1996). Recientemente también se ha demostrado que la alteración del repertorio peptídico de HLA-B27 induce la ausencia de artritis periférica en ratas transgénicas (Zhou et al., 1998). Esta es la evidencia más directa de un papel crítico de los péptidos de HLA-B27 en la patogenia de la enfermedad asociada con este antígeno. (iii) La sociación diferencial de los subtipos de HLA-B27 a EA. Este tema se desarrolla en detalle en el siguiente apartado. I.7.2. Distribución étnica y asociación a enfermedad de los subtipos de HLA-B27. Se conocen hasta el momento quince subtipos de HLA-B27 que difieren entre uno y siete residuos ubicados principalmente en el sitio de unión del péptido (Figura 6). Probablemente todos ellos han derivado por diferentes mecanismos evolutivos de B*2705 (López-Larrea et al., 1996). Sus distribuciónes étnicas y su asociación a enfermedad son variables (Tabla 1). Entre las poblaciones euro-caucasoides el subtipo mayoritario es B*2705, presente en el 90% de los individuos B27+. Prácticamente el 10% restante son B*2702. A su vez este es el subtipo predominante en judíos, donde representa el 60% de los individuos B27+ (GonzálezRoces et al., 1994). B*2709 es frecuente entre la población Sarda. B*2701, B*2708 y B*2710 son subtipos aparentemente minoritarios. B*2703 detectado inicialmente entre población negra de Norteamérica es originario de poblaciones del oeste africano. B*2704 y B*2706 se encuentran en poblaciones asiáticas, donde el primero es mayoritario. B*2707 es menos frecuente pero se encuentra también en poblaciones del sudeste asiático. B*2705, B*2702, B*2704(López-Larrea et al., 1995; Nasution et al., 1997; Ren et al., 1997) y B*2707 están claramente asociados a enfermedad. B*2706 y B*2709 (D’Amato et al., 1995) no están asociados a enfermadad en poblaciones donde otros subtipos si lo están aunque se han descrito dos casos de individuos B*2706 en China. B*2703 se ha encontrado en algún individuo con EA (González-Roces et al., 1997), sin embargo esta enfermedad es muy rara entre la poblacion del oeste de África, donde predomina este subtipo, incluso entre los individuos B*2705 de la misma población, lo que sugiere que

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probablemente están implicados otros factores genéticos protectores (Brown et al., 1997). Recientemente se ha encontrado co-segregación de EA con B*2708 en un estudio familiar (Armas et al., 1999). Los subtipos B*2701, B*2710, B*2711, B*2712. son subtipos poco frecuentes encontrados en individuos aislados, lo que impide un tratamiento estadístico adecuado para determinar su asociación a enfermedad. B*2713, B*2714 y B*2715 han sido descritos recientemente como nuevos subtipos. El primero es una variante cuya mutación aparece en la secuencia señal del exón 1 (Tabla 1).

α1 Subcavidad Subtipo B*2705 B*2701 B*2702 B*2703 B*2704 B*2706 B*2707 B*2708 B*2709 B*2710 B*2711 B*2712 B*2713 B*2714 B*2715

(a) (b) (c) (d) (e) (f) (g) (h) (i) (j) (k) (l) (m) (n) (ñ)

A

Asoc. a EA

# #

+ ¿? + + + − + + − ¿? ¿? ¿? ¿? ¿? ¿?

α2

C/F

C/F

C/F

C/F

α3

E C/F

D

D/E

C/F

E

59

69

70

71

74

77

80

81

82

83

97

113

114

116

131

152

211

Y − − H − − − − − − − − − −

A − − − − − − − − − − T − −

K − − − − − − − − − − N − −

A − − − − − − − − − − T − −

D Y − − − − − − − − − − − −

D N N − S S − S − − S S − −

T − I − − − − N − − − N − −

L A A − − − − − − − − − − −

L − − − − − − R − − − R − −

R − − − − −

N − − − − − S − − − S − −

Y − − − − − H − − − H − − −

H − − − − D N − − − N − − −

D

S − − − − − R − − − R − − −

V − − − E E − − − E − − − −

A − − − G G − − − n.d − n.d − −

G − − − G − −

− − − Y Y − H − Y − − −

Tabla 1: Residuos polimórficos entre los subtipos de HLA-B27. Los guiones indican identidad con el aminoácido presente en B*2705. (n.d; no determinado). Referencias: a: (Ezquerra et al., 1985; Moses et al., 1995). b: (Rojo et al., 1987a; Choo et al., 1986). c: (Vega et al., 1985a; Moses et al., 1995). d: (Rojo et al., 1987b; Choo et al., 1988). e: (Vega et al., 1985b; Rudwaleit et al., 1996). f: (Vega et al., 1986; Vilches et al., 1994a). g: (Choo et al., 1991). h: (Hildebrand et al., 1994). i: (Del Porto et al., 1994). j: (Fernández-Viña et al., 1996). k: (Hasegawa et al., 1997). l: (Balas et al., 1998). m: (Seurynck y Baxter-Lowe, 1998). n: (Hurley,C./ Nº acceso.AF072763-64, presenta los cambios L95W; N97T; V103L). ñ: (Van den Berg-Loonen E.M./ Nº acceso Y16637-38. Secuencia no publicada). # www.anthonynolan.com.

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INTRODUCCIÓN

69

71

74

70

77

80 82 81

83

59 97

6 11 11 3 11

4

152

131

Figura 6: Distribución de las posiciones polimórficas en los dominios α1 y α2 de la molécula de clase I HLAB27.

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II. OBJETIVOS

-25-

III. MATERIALES Y MÉTODOS

MATERIALES Y MÉTODOS

-27-

III. MATERIALES Y MÉTODOS

III.1. LÍNEAS CELULARES.

L

as células RMA-S pertenecen a una línea celular mutante derivada del linfoma de células T de ratón RBL-5 (H-2b) inducido por el virus de Rauscher (Ljunggren y Kärre, 1985). Estas células, son defectivas en proteínas TAP2 y muestran una baja expresión en

superficie de antígenos de clase I a 37ºC. Cuando se cultivan a temperaturas inferiores a 30ºC la expresión en membrana se incrementa, y expresan moléculas inestables de MHC de clase I vacías de péptido (Ljunggren et al., 1990b). La estabilidad, en estas condiciones, puede ser recuperada añadiendo péptido exógeno. En esta tesis se han utilizado diferentes transfectantes de subtipos y mutantes de HLA-B27 clonados previamente en el laboratorio. III.2. ANTICUERPOS MONOCLONALES (mAb). El anticuerpo ME1 (isotipo IgG1) (anti-HLA-B27+B7+B22) (Ellis et al., 1982), reconoce un

epítopo conformacional en el dominio α1 (El-Zaatari et al., 1990) que no es polimórfico entre los subtipos de HLA-B27, y que no se altera tras la unión del péptido (Smith et al., 1996a). Se obtuvo a partir de sobrenadante de cultivo del hibridoma secretor de dicho anticuerpo. Una vez verificada su correcta actividad, el sobrenadante se filtró y almacenó a –20ºC con 0,02% azida sódica. Como segundo anticuerpo, en citometría de flujo, se utilizó una dilución 1/100 de un mAb comercial fluoresceinado (FITC, Fluorescein Isothiocianate-conjugated rabbit F(ab’)2-anti-mouse IgG [H+L]) (Southern Biotechnology, Birmingham, Ala). III.3. SÍNTESIS PURIFICACIÓN Y CUANTIFICACIÓN DE PÉPTIDOS. III.3.1. Síntesis y purificación de péptidos y análogos no peptídicos. Los péptidos se sintetizaron en fase sólida mediante un sintetizador múltiple de péptidos AMS 422 utilizando la química F-moc. Tras la síntesis, se purificaron en HPLC de fase reversa midiendo su absorbancia a 210 y 280 nm., empleando un equipo Waters LC 625 provisto de una columna µ-Bondapack C18 (Waters) de 300 x 7,8mm. Se utilizó un gradiente de concentración creciente de acetonitrilo a un flujo de 2ml/min durante 60 minutos. con: (A): H2O +TFA 0,1%

MATERIALES Y MÉTODOS

-28-

(ácido trifluoroacético) y (B): acetonitrilo + TFA 0,1%. El gradiente, fue el siguiente: 0-5 minutos con 100% de (A), 5-40 min hasta un 40% de (B), de 40-45 hasta 60% de (B) de 45-50 hasta el 100% de (B). La pureza de los picos purificados se confirmó mediante HPLC analítico en fase reversa utilizando una columna Nova-Pack® C18 (Waters) 3,9 x 150mm a un flujo de 0,5ml/min y el mismo gradiente de acetonitrilo-TFA. La correcta masa molecular se determinó por espectrometría de masas mediante un espectrómetro VG Auto Spec. Los análogos no peptídicos utilizados en esta tesis, fueron sintetizados en el Laboratory for Organic Chermistry, Swiss Federal Institute of Technology. Zürich (Switzerland). Su cuantificación y control de pureza, se analizó igual que el resto de péptidos. III.3.2. Cuantificación. La cuantificación de los péptidos y su correcta composición, se determinó mediante un proceso previo de hidrólisis con ácido clorhídrico en atmósfera reductora de Nitrógeno, en presencia de un control estandard de norleucina, durante 24h a 110ºC. El análisis posterior se llevó a cabo mediante un analizador de aminoácidos (Beckman, Palo Alto, CA). Los péptidos se conservaron en agua Milli-Q a -70ºC a una concentración final 1mM. III.4. ENSAYO DE UNIÓN DE PÉPTIDOS.

El ensayo de unión de péptidos se realizó mediante un procedimiento cuantitativo (Galocha et al., 1996 / Anexo 2), basado en la estabilización, por péptidos añadidos exógenamente, de las moléculas vacías de HLA-B27 expresadas a 26ºC. Los transfectantes se incubaron a 26ºC durante 18-24 h, a una concentración de 106 células/ml en placas estériles de 96 pocillos de fondo en V (NUNC) en 100µl de RPMI 1640, 25mM HEPES y 10% de FBS (Suero bovino fetal) en ausencia de péptido. Transcurrida la incubación a 26ºC, las placas se lavaron dos veces con PBS estéril y se les añadió el péptido diluido en medio RPMI 1640, 25mM HEPES sin suero, para obtener concentraciones finales comprendidas entre 10-4M hasta 10-9M. Posteriormente las células se incubaron durante una hora a 26ºC y, tras este periodo, a 37ºC durante 2h o 4h dependiendo del subtipo: B*2701, B*2702, B*2703, B*2704 y los mutantes Y74, A81, D114 y E152 se incubaron durante 2h. B*2705, B*2706, S77, Y116, D114Y116 se incubaron durante 4h. Estos tiempos se eligieron de forma que la expresión de HLA-B27 era claramente superior a la expresión basal a 37ºC, pero en los que en presencia de péptido la disociación y pérdida de expresión en superficie predominase

-29-

MATERIALES Y MÉTODOS

sobre la asociación e inducción de la expresión.

III.4.1. Análisis por citometría de flujo. Las células se lavaron dos veces con PBS para eliminar cualquier traza de péptido en el sobrenadante y se incubaron nuevamente durante 30 minutos a 4ºC con 30-50 µl de anticuerpo monoclonal ME1. Tras este período, se lavaron otras dos veces con PBS estéril y se añadieron de 30-50µl del segundo anticuerpo monoclonal fluoresceinado, dejando transcurrir otros 30 minutos a 4ºC. Transcurrida la segunda incubación, se lavaron dos veces con PBS estéril. y se fijaron con 100µl de paraformaldehído previamente a su análisis en un citómetro de flujo EPICS Profile II Coulter donde se midió la fluorescencia lineal para un total de 5000. células. III.4.2. Cálculo de la unión de péptidos. La media de la fluorescencia lineal asociada a la expresión de HLA-B27 se representó en función de las diferentes concentraciones de péptido. El análisis cuantitativo y comparativo de la unión de los diferentes péptidos se realizó mediante el programa Origin MicroCal Software Inc. Tras calcular la concentración molar de péptido a la cual se producía el 50% de la fluorescencia máxima (C50). La concentración molar requerida de cada péptido para alcanzar el valor de fluorescencia C50 del péptido utilizado como referencia o control, se denominó EC50. La unión relativa se definió como la relación molar entre los valores EC50 de los péptidos comparados. Los intervalos de afinidad se escogieron en función de los valores de afinidad obtenidos en los ensayos de unión con los péptidos presentados in vivo. Así las afinidades se definieron como sigue: (Afinidad alta: <5µΜ.), (Afinidad intermedia: 6µM-50µM ) y (Afinidad baja: >50µM). La afinidad de péptidos mayor de 100µM no se midió en este ensayo, y refleja una unión marginal o nula.

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IV. RESULTADOS

RESULTADOS

-33-

IV. RESULTADOS IV.1. EFECTO DEL POLIMORFISMO DE HLA-B27 SOBRE LA ESPECIFICIDAD DE UNIÓN DE PÉPTIDOS.

C

on la finalidad de determinar las bases moleculares que determinan el solapamiento de repertorios peptídicos entre subtipos de HLA-B27, el estudio se centró en el análisis de la especificidad peptídica de: (i) B*2704 y B*2706, (ii) B*2701 y (iii), B*2703.

B*2704 y B*2706 están relacionados estructuralmente pero desigualmente asociados a

enfermedad. B*2701 y B*2702 difieren de B*2705 en mutaciones distintas pero localizadas en la misma región de la molécula. El polimorfismo de B*2703, afecta a una interacción conservada con el extremo peptídico N-terminal. Las diferencias entre estos subtipos se analizaron comparando las afinidades de unión in vitro de ligandos naturales sintéticos de B*2705 (05.Pi) (Jardetzky et al., 1991) y B*2702 (02.Pi) (Rötzschke et al., 1994). Las bases moleculares de estas diferencias se analizaran con mutantes que mimetizaban los cambios puntuales entre los subtipos, y mediante análogos peptídicos a los que se cambia en los residuos principales de anclaje PΩ o P2. IV.1.1. Unión de péptidos a HLA-B*2705, B*2704 y B*2706. Modulación de la especificidad por el residuo peptídico C-terminal. (Anexo 1) B*2704 y B*2706, son los dos subtipos HLA-B27 restringidos a poblaciones asiáticas, muy diferentes antigénicamente de B*2705 (López et al., 1994). B*2706 no está asociado a enfermedad en poblaciones donde B*2704 si lo está. B*2704 difiere de B*2705 en dos cambios: D77S y V152E. B*2706 tiene además dos cambios adicionales a los de B*2704, H114D y D116Y. Ambos subtipos tienen además el cambio A211G, que por su localización, en el dominio α3, no afecta a la unión del péptido. Tanto los ligandos (05.Pi) como (02.Pi), se unieron a B*2705 con alta afinidad (EC50 ≤5µM), (Tabla 2), lo que sugiere que el repertorio de B*2705 podría englobar una parte del repertorio natural de B*2702. El estudio con los análogos peptídicos en P2 mostró un descenso de la afinidad de unión,

RESULTADOS

-34-

indicando que este anclaje era esencial para mantener la buena unión del péptido, sin embargo a diferencia de lo que ocurre con los análogos de Ala2, el residuo Gln2 funcionaba como un residuo subóptimo permitiendo una unión significativa, y por lo tanto compatible con la presencia de este residuo entre los ligandos naturales de B*2705 (Tabla 3A). La contribición del residuo P9 fue variable entre los diferentes análogos. Así, Lys en 05.P2 era mejor que en 05.P6 mientras que Tyr era equivalente a Arg y Leu en 02.P4 y 02.P6, y también a Ala en 02.P4 pero no en 02.P6 (Tabla 3B). Esto indica que la tolerancia de determinados residuos está influida por el resto de la secuencia del péptido. El estudio de la contribución de los diferentes residuos debe realizarse con análogos que eviten, en lo posible, la interferencia de otras posiciones (ver Anexo 5). B*2704, unió eficientemente péptidos con residuo C-terminal alifático (Leu) o aromático (Tyr) (EC50≤5µM) y peor aquellos con residuo básico (Tabla 2). Los análogos con Gln2 de péptidos con residuos C-terminales alifáticos (Leu) se unieron a este subtipo mejor que a B*2705, indicando que el anclaje C-terminal hidrofóbico es más fuerte que en B*2705. En cambio en 05.P6Q2 no se unió, lo que significa que la unión de Lys C-terminal es más débil que en B*2705 (Tabla 3B). La unión de los análogos de 02.P4 y 02.P6 con L9 (Tabla 3B) fue superior a la de los péptidos correspondientes, indicando que Leu es mejor residuo C-terminal que Tyr para la unión a B*2704. B*2706 unió eficientemente péptidos con residuos C-terminales apolares (Leu, Phe), pero no aquellos con Tyr o residuos básicos (Tabla 2). La unión relativa de los análogos con Gln2, que fue buena para péptidos con Leu y Phe y mala para Lys o Tyr, confirmó estas preferencias (Tabla 3A) La unión de análogos con L9 fue claramente superior que en los péptidos con Tyr C-terminal (02.P4, 02.P6) lo que confirma las preferencias de B*2706 por Leu respecto a Tyr. Estos resultados indican que B*2704 y B*2706 difieren de B*2705 sobre todo en su peor tolerancia por residuos C-terminales básicos. Además la diferencia fundamental entre B*2704 y B*2706, reside en la menor aceptación por este último de residuos con Tyr C-terminal. IV.1.1.1. Efecto de las pérdidas y ganancias de residuos cargados en las subcavidades E/C/F. El uso de mutantes que reproducen los cambios en B*2705 y B*2706, permitió el análisis del papel que juegan las posiciones polimórficas que diferencian a estos subtipos. Estas mutaciones determinan las propiedades electrostáticas de las subcavidades donde interacciona el péptido. La eliminación de una carga negativa en la cavidad C/F, como ocurre con los mutantes S77 y

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RESULTADOS

Y116 no impidió la unión eficiente de los ligandos naturales, independientemente del residuo Cterminal (Tabla 2). Además, en general, los péptidos con residuos C-terminales apolares se unieron mejor a los dos mutantes que a B*2705. Una característica importante del mutante Y116 fue el aumento notable de su preferencia por Leu como residuo C-terminal respecto a Tyr, como se apreció con los análogos de 02.P4 y 02.P6.(Tabla 3B). D114 y E152, al contrario que las mutaciones anteriores, suponen la ganancia de una carga negativa. La unión de los ligandos naturales a estos mutantes mostró un empeoramiento general de la unión relativa de los péptidos respecto a la unión a B*2705 (Tabla 2). La doble mutación D114Y116, supone un balance neto de cargas neutro. Este mutante unió bien los péptidos con residuos C-terminales tanto básicos, como alifáticos o aromáticos, y en general las afinidades de unión al doble mutante se mostraron similares a las del mutante Y116, indicando el efecto compensador de esta mutación sobre los efectos adversos de la mutación D114.

PÉPTIDO

SECUENCIA

B*2705 B*2704 B*2706

S77

Y116

E152

D114

DY

05.P2 05.P6

RRIKEIVKK GRIDKPILK

0,8 2

10 20

100 70

0,5 1

0,1 1

6 20

60 5

0,1 0,3

05.P5 05.P8

RRSKEITVR KRFEGLTQR

2 2

10 20

30 40

1 0,6

3 2

20 >100

40 90

3 2

05.P10 02.P6

RRISGVDRY KRGILTLKY

3 4

5 2

40 40

0,8 0,3

5 5

>100 30

40 10

4 2

02.P2 02.P3

GRLTKHTKF RRFVNVVPTF

4 2

4 2

4 1

0,9 0,5

1 0,4

5 3

8 4

0,4 0,5

05.P1

RRYQKSTEL

1

1

0,5

1

0,1

5

20

0,2

Tabla 2: Unión de ligandos naturales de B*2705 y B*2702, a subtipos y mutantes de HLA-B27. La unión a cada variante de HLA-B27 está expresada en valores de EC50 (µM) que indica la concentración requerida de péptido para obtener la mitad de la fluorescencia máxima (C50), asociada a HLA-B27 unido al péptido de máxima afinidad entre los péptidos de las series 05.Pi y 02.Pi. Los péptidos con EC50 ≤ 5µM se considera que tienen alta afinidad por ser este el rango en el que se unen los péptidos naturales presentados por B*2702 y B*2705 respectivamente. EC50 ≥ 50 µM indica baja afinidad, 5 µM ≤ EC50 ≤ 50 µM afinidad intermedia, EC50≥100µM, carencia de unión.

RESULTADOS

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Tabla 3A Péptido

Secuencia

B*2705 B*2704 B*2706

S77

Y116

E152

D114

DY

05.P1 05.P1Q2 05.P1A2

RRYQKSTEL -Q------L -A------L

1 (1) 10 90

1 (2) 2 4

1 (0,1) 1 5

1 (2) 0,5 3

1 (0,05) 6 60

1 (3) 7 -

1 (20) 5 -

1 (0,4) 2,5 7,5

05.P4 05.P4Q2 05.P4A2

RRWLPAGDA -Q------A -A------A

1 (5) -

1 (7) -

1 (9) 8

1 (1) 40 60

1 (2) 20 20

1 (30) -

1 (20) 2 2

1 (3) 3 13

05.P6 05.P6Q2 05.P6A2

GRIDKPILK -Q------K -A------K

1 (0,9) 11 -

1 (12) -

1 (50) -

1 (1) 30 -

1 (1) 6 -

1 (26) -

1 (4) 10 -

1 (0,1) 2 -

02.P2 02.P2Q2 02.P2A2

GRLTKHTKF -Q------F -A------F

1 (4) 18 -

1 (4) 5 -

1 (2) 4 40

1(0,9) 6 8

1 (0,6) 5 17

1 (6) -

1 (6) -

1 (0,2) 2,5 20

02.P4 02.P4Q2 02.P4A2

KRYKSIVKY -Q------Y -A------Y

1 (3) 20 -

1 (6) 5 -

1 (2) 25 -

1 (0,5) 10 -

1 (1) 30 -

1 (10) 4 -

1 (8) -

1 (1) 30 -

02.P6 02.P6Q2 02.P6A2

KRGILTLKY -Q------Y -A------Y

1 (1) -

1 (5) 10 -

1 (40) -

1 (0,2) 40 -

1 (3) 13 -

1 (50) -

1 (20) -

1 (5) -

Tabla 3B

Péptido

Secuencia

B*2705 B*2704 B*2706

Y116

D114

05.P6 05.P6A9

GRIDKPILK -R------A

1 (0,9) 8

1 (12) 0,6

1 (50) 0,04

02.P2 02.P2R9

GRLTKHTKF -R------R

1 (4) 1

1 (4) 5

1 (2) 30

02.P4 02.P4R9 02.P4L9 02.P4A9

KRYKSIVKY -R------R -R------L -R------A

1 (3) 1 0,7 1

1 (6) 1 0,3 1

1 (2) 0,4 0,2 0,4

1 (1) 5 0,01 0,04

1 (8) 0,4 0,4 1

02.P6 02.P6R9 02.P6L9 02.P6A9

KRGILTLKY -R------R -R------L -R------A

1 (1) 1 0,9 20

1 (5) 2 0,6 1

1 (40) 0,02 0,08

1 (3) 3 0,002 0,2

1 (20) 0,4 0,3 -

La unión relativa está expresada como el cociente entre el EC50 del correspondiente análogo y el C50 del ligando natural. Los valores de C50 (µM) del péptido sin cambios aparecen indicados entre paréntesis. Los guiones indican no unión del péptido.

Tabla 3: Efecto de los análogos en P2 (Tabla 3A) y P9 (Tabla 3B) en la unión relativa a subtipos y mutantes.

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RESULTADOS

IV.1.2. Unión de péptidos a B*2701 y B*2702. Efecto del polimorfismo sobre la especificidad de los residuos en P2. (Anexo 2) En este estudio, siguiendo una estrategia similar a la del apartado anterior, se determinó la especificidad de unión de péptidos in vitro a B*2701 y B*2702. B*2701 es un subtipo poco frecuente, cuya asociación a enfermedad se desconoce; se diferencia de B*2705 en los cambios D74Y, D77N y L81A. El primero es un cambio único entre los subtipos conocidos de HLA-B27. B*2702, que sí está asociado a enfermedad, se diferencia de B*2705 en los siguientes cambios: D77N, T80I y L81A. La unión de los péptidos 05.Pi y 02.Pi mostró que tanto B*2701 como B*2702 presentaban preferencias similares por residuos C-terminales alifáticos (Leu) y aromáticos (Phe y Tyr). Los residuos C-terminales básicos estaban desfavorecidos en la unión a B*2701 pero B*2702 también unía péptidos con Arg9 (05.P5, 05.P8) con afinidades compatibles con su posible presentación in vivo (Tabla 4). Estos resultados sugieren que B*2701 y B*2702 comparten con B*2705 una parte del repertorio peptídico, que incluye sobre todo residuos C-terminales alifáticos o aromáticos. Los análogos con Gln2 de los péptidos con residuos C-terminales básicos se unieron mal a B*2702, pero esos mismos análogos de péptidos con Leu, Tyr o Phe C-terminal (05.P1Q2, 02.P4Q2, 02.P6Q2) se unieron de forma similar a los péptidos naturales, mostrando una mejor unión relativa a B*2702 que a B*2705 (Tabla 5A). Estos resultados sugieren que los residuos alifáticos y aromáticos, están más favorecidos en B*2702 que en B*2705, y que los péptidos con Gln2 podrían formar parte del repertorio natural de B*2702, aunque no han sido aún encontrados. Todos los análogos con Gln2, independientemente del residuo C-terminal, se unieron a B*2701 con una eficiencia similar a la del péptido natural, indicando que las contribuciónes de la Arg2 y de la Gln2 en B*2701 son equiparables. La preferencia, tanto de B*2701 como de B*2702 por Leu, Phe y Tyr C-terminal, se confirmó mediante los análogos con cambios en esta posición. La unión relativa de los análogos con Leu C-terminal (02.P4L9 y 02.P6L9, originalmente Tyr9) mostraron una eficiencia de unión similar, indicando que tanto Tyr como Leu son igualmente adecuados en ambos subtipos. Sin embargo, la peor unión a B*2701 de los análogos con Ala9 de esos mismos péptidos, sugiere que los residuos C-terminales Tyr y Leu contribuyen más fuertemente a la unión en B*2701 que en B*2702 (Tabla 5B). En general la unión de los péptidos naturales al mutante Y74 fue buena (Tabla 4), por

RESULTADOS

-38-

tanto, la mala unión de algunos péptidos a B*2701 no era debida a esta mutación. Por el contrario, la unión de los mismos péptidos al mutante A81 fue bastante peor, indicando que esta mutación es probablemente responsable de la deficiente unión de muchos de los péptidos a B*2701. El hecho de que algunos de los péptidos se unían bien a B*2702, que también posee el cambio A81, sugiere la existencia de efectos compensadores de otros cambios, posiblemente I80, en este subtipo (Tabla 4). Al igual que con B*2701, los análogos con Gln2 se unieron bien al mutante Y74, indicando que la preferencia de B*2701 por Gln2 es debida al efecto de esta mutación (Tabla 5A). Debido a que la posición Y74 se localiza fuera de la subcavidad B, este resultado indica que la especificidad de esta subcavidad es modulada por polimorfismo existente fuera de ella. Confirmando los resultados de unión in vitro, la secuenciación de los respectivos pooles de péptidos demostró la presencia, tanto de Arg2 como de Gln2 entre los péptidos presentados in vivo por B*2701 y por el mutante Y74 (Anexo 2).

Péptido

Secuencia

B*2705 B*2702 B*2701

Y74

A81

05.P1 05.P2 05.P3 05.P11 05.P6 05.P5 05.P7 05.P8 05.P10 05.P4 05.P9

RRYQKSTEL RRIKEIVKK RRVKEVVKK ARLFGIRAK GRIDKPILK RRSKEITVR FRYNGLIHR KRFEGLTQR RRISGVDRY RRWLPAGDA RRFTRPEH

1 0,8 2 5 2 2 4 2 3 4 20

8 20 50 >100 20 20 6 4 8 6 40

5 >100 >100 >100 >100 >100 40 30 4 >100 >100

0,7 0,8 2 3 1 1 0,6 0,4 2 3 20

10 9 20 80 20 80 20 30 40 10 70

02.P3 02.P2 02.P4 02.P6

RRFVNVVPTF GRLTKHTKF KRYKSIVKY KRGILTLKY

2 4 4 4

1 2 2 1

0,9 10 1 0,6

0,5 3 1 3

2 9 20 7

Tabla 4: Unión de ligandos naturales de B*2705 y B*2702 a subtipos y mutantes de HLA-B27. Se indican valores de EC50 (µM).( ver pie de la tabla 2)

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Tabla 5: Unión relativa de varios análogos peptídicos con cambios en las posiciones P2 (Tabla 5A) y P9 (Tabla 5B) a los B*2705, B*2702 y B*2701 y a los mutantes Y74 y A81. La unión relativa está expresada como el cociente del entre el EC50 correspondiente análogo y el C50 del ligando natural. Entre paréntesis aparecen Los valores de C50 (µM) del péptido sin cambios. Los guiones indican no unión del péptido.

Tabla 5A

Tabla 5B

RESULTADOS

Péptido

Secuencia

B*2705 B*2702 B*2701

Y74

A81

1 (1) 0,8 20

1 (12) -

1 (2) 2 -

1 (20) 4 -

05.P1 05.P1Q2 05.P1A2

RRYQKSTEL -Q------L -A------L

1 (1) 10 90

1 (4) 1 15

05.P6 05.P6Q2 05.P6A2

GRIDKPILK -Q------K -A------K

1 (0,9) 11 -

1 (10) -

05.P7 05.P7Q2

FRYNGLIHR -Q------R

1 (2) 5

1 (5) 8

1 (20) 1,5

1(21) 2

1 (24) 3

02.P2 02.P2Q2 02.P2A2

GRLTKHTKF -Q------F -A------F

1 (4) 18 -

1 (4) 7,5 -

1 (6) 0,5 -

1(0,8) 2,5 -

1 (9) -

02.P3 02.P3Q2 02.P3A2

RRFVNVVPTF -Q-------F -A-------F

1 (2) 3 35

1 (1) 3 20

1 (2) 1 2,5

1(1) 1 20

1 (3) 10 -

02.P4 02.P4Q2 02.P4A2

KRYKSIVKY -Q------Y -A------Y

1 (3) 20 -

1 (4) 1 15

1 (2) 0,4 2,5

1 (1) 0,7 20

1 (10) 9 -

02.P6 02.P6Q2 02.P6A2

KRGILTLKY -Q------Y -A------Y

1 (1) -

1 (1) 2 80

1 (0,7) 0,9 29

1 (1) 2 -

1 (15) -

Péptido 05.P6 05.P6A9

GRIDKPILK -R------A

1 (0,9) 8

1 (10) 0,4

02.P2 02.P2R9

GRLTKHTKF -R------R

1 (4) 1

1 (4) 15

02.P3 02.P3R10 02.P3A10

RRFVNVVPTF -R-------R -R-------A

1 (2) 2 2

02.P4 02.P4R9 02.P4L9 02.P4A9

KRYKSIVKY -R------R -R------L -R------A

02.P6 02.P6R9 02.P6L9 02.P6A9

KRGILTLKY -R------R -R------L -R------A

Secuencia

1 (5) 0,8 1,4

B*2705 B*2702 B*2701

Y74

A81

1 (2) 2

1 (20) 1,5

1 (6) -

1(0,8) 2,5

1 (9) 1

1 (1) 4 2

1 (2) 35 3,5

1(1) 2 3

1 (3) 3 7

1 (3) 1 0,7 1

1 (4) 12,5 1 2,5

1 (2) 10 1,5 40

1 (1) 1 0,6 1

1 (10) 1 0,3 1

1 (1) 1 0,9 20

1 (1) 20 1 10

1 (0,7) 1,3 -

1 (1) 2 0,7 10

1 (15) 1,3 0,2 -

RESULTADOS

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IV.1.3. Unión de péptidos a B*2703. Papel del polimorfismo de la subcavidad A. (Anexo 3) B*2705 y B*2703 se diferencian exclusivamente en el cambio Y59H, localizado en la subcavidad A. Este residuo está implicado en la estabilización del extremo N-terminal del péptido, y está conservado entre los subtipos de HLA-B27 a excepción de B*2703. Para determinar los efectos de esta

Péptido

Secuencia

(1) (2) (3)

RRYQKSTEL ARYQKSTEL RQYQKSTEL

mutación en la interacción con péptido, ésta se estudió bajo dos puntos de vista. En primer lugar, mediante un ensayo de unión

Unión relativa B*2703 B*2705 1 (2.10-6) 1,5 >>100

1 (2.10-6) 2 10

in vitro se determinaron las eficiencias de unión a B*2705 y B*2703 de tres péptidos: (1) RRYQKSTEL, un ligando natural de ambos subtipos (Jardetzky et al, 1991; Boisgérault et al, 1996.) y dos análogos del mismo,

(2)

ARYQKSTEL

y

(3)

RQYQKSTEL (Tabla 6).

Tabla 6: Afinidades de unión de tres péptidos a los subtipos B*2705 y B*2703. Los valores correspondientes al C50 (µM) del péptido (1), que aparece entre paréntesis, indican la concentración en la cuál se alcanza la mitad de la máxima fluorescencia, determinada mediante citofluorimetría. La unión relativa de los análogos, indica el exceso molar de péptido necesario para alcanzar el valor de C50

Los resultados indican que el cambio de Arg por Ala en P1 tiene un efecto limitado y cualitativamente similar sobre la unión a ambos subtipos. Sin embargo, el cambio Arg por Gln en P2 tiene un efecto mucho más dramático sobre la unión a B*2703 que a B*2705. Esto muestra que el cambio Y59H en B*2703, además de los efectos en la subcavidad A afecta a las interacciones en la subcavidad B, aumentando la preferencia por Arg2 con relación a Gln2. Los efectos de los cambios peptídicos sobre su unión a ambos subtipos, se analizaron mediante una simulación de dinámica molecular basada en la estructura cristalográfica de HLAB27. Esta técnica permite predecir varios parámetros moleculares (distancias entre ligando y el centro de masas de la proteína, fluctuaciones atómicas, áreas accesibles y no accesibles del ligando, frecuencias de formación de enlaces de hidrógeno) que ayudan a interpretar las características de la unión a HLA-B27 observadas experimentalmente (Rognan et al., 1994). IV.1.3.1. Propiedades dinámicas vs afinidad de unión de péptidos. Mediante la determinación de las variaciones en las distancias entre el péptido y un centro de masas teórico de la molécula de HLA-B27, puede obtenerse una medida indirecta de la

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RESULTADOS

movilidad del péptido y, por tanto, de su estabilidad. Definiendo una serie de distancias: d1: Distancia proteína-péptido; d2: proteína-(P1, P2, P3), d3: proteína-(P4, P5, P6, P7 y P8); d4: Distancia entre la subcavidad A y el residuo P1, d5: Distancia subcavidad B-residuo P2; d6: Distancia subcavidad D-residuo P3 y d7: Distancia subcavidad F-residuo P9, la modelización permite determinar lo que sucede en la interacción de los diferentes péptidos a ambos subtipos. Cuantitativamente, la parte central del péptido expuesta al TCR (residuos P4-P8) sufrió un incremento de las distancias, similar en los tres péptidos unidos a ambos subtipos (Anexo 3, Tabla 2: d3). El análisis de las distancias d4-d7 entre los anclajes y sus subcavidades complementarias A, B, D y F mostró que el péptido (3), tanto unido a B*2703 como a B*2705 experimentaba una repulsión localizada en el residuo P9 (d7), muy alejada del lugar de la mutación en la subcavidad A, así como una mayor distancia d5 para el complejo RQYQKSTELB*2703. La distancia d4, era dependiente del residuo presente en P1, aunque para un mismo P1 siempre fue mayor en el subtipo B*2703. Por último, las distancias d6 fueron más variables demostrando la flexibilidad de ésta (Anexo 3, Tabla2). Estos datos indican que la mutación Tyr→His59 desestabiliza la subcavidad A, de modo que en estas circunstancias, un mal anclaje en P2 causa una inestabilidad global del péptido en varias posiciones, y de forma especial en su extremo C-terminal. IV.1.3.2. Fluctuaciones atómicas, áreas accesibles y no accesibles. Como era de esperar, la parte central del péptido expuesta al TCR mostró mayores fluctuaciones que las posiciones de anclaje del péptido. En concordancia con los valores de afinidad de unión in vitro, la comparación de las movilidades atómicas de los péptidos (1) y (2) unidos a ambos subtipos no mostró diferencias significativas, pero la flexibilidad del péptido (3) se incrementó, especialmente en el complejo de menor afinidad RQYQKSTEL-B*2703 (Anexo 3: Figura 3). La magnitud de las fluctuaciones en el residuo P9, indican que cuando el anclaje en P2 es débil, la inestabilidad se distribuye a otras posiciones de anclaje incluída, de forma prominente, la posición C-terminal. En consonancia con el resultado anterior, el análisis de las áreas accesibles frente a las no accesibles, nuevamente mostró que la mayor accesibilidad de P9, se daba en el complejo RQYQKSTEL-B*2703 (Anexo 3: Figura 4). Sin embargo, a pesar de la flexibilidad que mostró la

Gln2 (Anexo 3: Figura 3), este residuo permaneció inaccesible sugiriendo que, aunque la Gln2 no es por si misma un buen residuo de anclaje, aún conserva interacciones en la subcavidad B.

RESULTADOS

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IV.1.3.3. Análisis cualitativo y cuantitativo de los puentes de hidrógeno La estabilidad de los puentes de hidrógeno establecidos entre HLA-B27 y el péptido, se determinó mediante el cálculo de la frecuencia de formación de dichos enlaces. Se consideró como un puente de hidrógeno fuerte aquél con una frecuencia mayor del 50%, medio cuando la frecuencia estaba entre el 25% y el 50%, y débil si era inferior al 25%. Con estas premisas, el número, distribución y frecuencia de los puentes de hidrógeno permitió distinguir cualitativa y cuantitativamente, los péptidos (1) RRYQKSTEL, (2) ARYQKSTEL y (3) RQYQKSTEL (Anexo 3: Figura 5). La modelización predijo un número de 25 enlaces para los péptidos (1) y (2) unidos a ambos subtipos, y de la mitad cuando el péptido unido era (3). Asimismo, la distribución de los enlaces débiles y fuertes se correspondió con las afinidades de unión in vitro, de forma que los complejos formados por los péptidos (1) y (2) mostraron un número similar de enlaces fuertes en ambos subtipos frente al reducido número de enlaces medios o fuertes encontrados para el péptido (3). Estos resultados indican que la afinidad de unión de un péptido está directamente relacionada con la cantidad y calidad de los enlaces de hidrógeno que establece el péptido con diferentes residuos de la molécula de clase I.

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RESULTADOS

IV.2. RELACIÓN ENTRE LA UNIÓN DE PÉPTIDOS Y LA SELECCIÓN DE EPÍTOPOS VIRALES POR CÉLULAS T. (Anexo 4)

El polimorfismo de las moléculas de clase I tiene una función clave en el reconocimiento por las células T de los complejos MHC-Péptido, puesto que determina la presentación o no de un péptido antigénico, modula su afinidad y estabilidad de unión, y puede alterar la conformación final del epítopo reconocible por el TCR. En este estudio se trató de determinar el papel que juega el polimorfismo de HLA-B27, en la presentación y en el reconocimiento por células T de péptidos derivados del virus de Epstein-Barr (EBV), que son inmunogénicos sólo en el contexto de determinados subtipos de HLA-B27. IV.2.1. Unión de péptidos virales a diferentes subtipos de HLA-B27, y su relación con la inmunogenicidad. Se estudió la unión de tres péptidos derivados del EBV, restringidos por diferentes subtipos de HLA-B27. EBNA3C (258-266) (Brooks et al., 1993) es restringido por los subtipos B*2705 / B*2702 / B*2704; LMP2 (236-244) es restringido por B*2704 (Brooks et al., 1993) y EBNA3B (243-253) es restringido por B*2702 (Brooks et al., 1998) (Tabla 7). Aunque todos se

unieron a los tres subtipos, EBNA3B se unió mejor a B*2705 que a B*2702, y LMP2 se unió con igual afinidad a B*2705 y B*2704. Por tanto, no existe una correlación entre la eficiencia de unión in vitro de un péptido viral a un subtipo de HLA-B27, y su inmunogenicidad en el contexto de dicho subtipo. La unión relativa tampoco se correspondió con sus patrones de restricción; así en el caso de LMP2 la unión a B*2702 fue 30 veces mejor que la unión de EBNA3B y 7,5 veces mejor que la unión de EBNA3C a B*2705 (Tabla 7). Estos resultados

indican que la unión y la inmunogenicidad de un péptido, están determinadas por factores distintos al de su afinidad por el subtipo HLA que actúa como elemento de restricción. Los valores entre paréntesis corresponden al C50 µM del péptido que mejor unión presentaba (LMP2). El resto de valores corresponde al exceso molar necesario de péptido al necesario para alcanzar el valor de C50 .

Péptido

Restricción

LMP2 EBNA3C EBNA3B

B*2704 B*2705 / 02 / 04 B*2702

Secuencia RRRWRRLTV RRIYDLIEL RRARSLSAERY

B*2705

B*2702

B*2704

1 (0,4 µM) 1 (1 µM) 1 (0,4 µM) 7,5 3 12,5 15 30 50

Tabla 7: Unión de péptidos de EBV a tres subtipos de HLA-B27 que actúan como elementos de restricción.

RESULTADOS

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La unión de los tres péptidos virales a otros subtipos de HLA-B27 (B*2705, B*2703 y B*2706) fue buena en general (Anexo 4, Tabla 3), lo que indica que el polimorfismo entre los subtipos B*2701-B*2706, no afecta críticamente a la unión de los péptidos estudiados. Sin embargo, para LMP2 las diferencias de afinidad por diferentes subtipos se hicieron más patentes mediante el uso de análogos con Ala2 (Anexo 4, Tabla4). La unión relativa de LMP2A2 fue más baja para B*2705 / 03 / 01 que para su elemento de restricción (B*2704) y para B*2706 y B*2702. Este resultado confirma que la inmunogenicidad de LMP2, exclusivamente en B*2704 no está relacionada con su mayor afinidad por este subtipo. IV.2.2. Reconocimiento de péptidos de EBV por CTLs restringidos por HLA-B27. En estos experimentos se analizó la capacidad de CTLs específicos de péptido, y restringidos por un subtipo determinado, para reconocer dicho péptido en el contexto de otros subtipos. La capacidad de los tres péptidos de EBV, para ser reconocidos por diferentes CTLs restringidos por B*2705, B*2704 y B*2702 se resume en la Tabla 8. De 10 clones restringidos por B*2705 específicos de EBNA3C, tres reconocieron al péptido también en el contexto de B*2702. De igual forma, cuatro de los cinco clones restringidos por B*2702 contra el mismo péptido lo reconocieron en el contexto de B*2705, pero ninguno de los quince lo reconocieron unido a B*2704. Los clones restringidos por B*2702 específicos de EBNA3B, también reconocían al péptido, unido tanto a B*2702 como a B*2705 pero no a

B*2704. De los cuatro clones restringidos por B*2704 específicos de LMP2, ninguno reconoció al péptido unido ni a B*2705 ni a B*2702. Estos resultados muestran que B*2705 y B*2702, pueden actuar como elementos de restricción equivalentes, al menos para algunos CTLs. Sin embargo, B*2704 muestra un grado mucho mayor grado de disparidad funcional. IV.2.3. El motivo Arg2, anclaje principal de los péptidos unidos a HLA-B27, no es esencial para mantener la capacidad antigénica del péptido. En estos experimentos se analizó el papel del residuo principal de anclaje de los péptidos en HLA-B27, en mantener la capacidad antigénica del epítopo viral. El análisis realizado con el péptido EBNA3C y sus análogos con Gln2 y Ala2 mostró diferencias en la afinidad de unión a los subtipos que actuan como elementos de restricción para este péptido, en función del residuo presente en P2 y del subtipo (Figura 7A). No obstante, los CTLs restringidos por B*2705 reconocieron tanto al péptido como a sus análogos. Los CTLs

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RESULTADOS

restringidos por B*2702 reconocieron al análogo con Ala2 mucho peor. El mismo análisis realizado con los análogos del péptido EBNA3B y sus análogos en P2 mostró una unión similar del análogo con Gln2 a B*2702, sin embargo el análogo con Ala2 aunque se unió bastante peor, fue reconocido con igual eficacia por los CTLs restringidos por B*2702 específicos de EBNA3B. (Figura 7B). Estos resultados mostraron que el residuo de anclaje principal Arg2 es un requisito fundamental para obtener una buena unión los péptidos a HLA-B27, pero no es esencial para mantener la estructura antigénica del péptido.

RESULTADOS

Tabla 8: Reconocimiento por CTLs restringidos por HLA-B27 de péptidos derivados de EBV. Los valores indican el % de lisis específica a una relación Efector:Diana de 4:1. Las células diana se incubaron durante 30 minutos con una concentración de péptido 10-6M. En todos los casos el fondo fue inferior al 5%. En la columna derecha aparece el número de clones que tenían el mismo patrón de reactividad. En negrita y para mayor claridad, se marcan los casos en los que la reactividad es claramente positiva.

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Clon

Donante

Célula Diana

Nº clones

B*2705

B*2702

B*2704

B*2705 B*2705

65 51

3 60

2 1

7 3 Total 10

B*2702 B*2702

9 44

55 53

0 0

1 4 Total 5

B*2702 B*2702

72 50

76 64

0 0

B*2704

0

3

35

EBNA3C RTc10 RTc37 EBNA3C LYc39 Kor c69 EBNA3B LYc40 NWc20

Total 4

LMP2 DHc21

4 Total 4

Figura 7:

B/ Unión del péptido EBNA3B y sus análogos al subtipo que actúa como su elemento de restricción, (B*2702). A la izquierda, se muestra la lisis de una línea celular linfoblastoide autóloga por el clon restringido y específico de péptido LYc29. Relación E:D = 4:1.

SECUENCIA

B*2702

EBNA3C

RRIYDLIEL

3

3

5

EBNA3CA2

-A-------

−

−

−

EBNA3CQ2

-Q-------

20

100

−

80

% de lisis específica

A/ Arriba; unión del péptido EBNA3C y sus análogos, a los tres subtipos que actúan como sus elementos de restricción. Debajo, a la izquierda, lísis de una línea celular linfoblastoide autóloga, por un CTL específico del péptido, provenientes de un donante B*2702+(LYc25), y sensibilizada a varias concentraciones con los péptidos anteriores. Relación Efector : Diana=4:1. A la derecha un experimento similar en el que diana y efector (Alc12) eran de un individuo B*2705.+ Relación E:D=2:1.

PÉPTIDO

A

EBNA 3C EBNA 3CA2 EBNA 3CQ2 SIN PÉPTIDO

60

B*2704

100

AL c12

80 60 40

20

20

0

0 -11

B

LY c25

B*2705

40

10

% de lisis específica

Unión y reconocimiento de dos péptidos virales y sus análogos en P2 por CTLs específicos de péptido.

-10

10

-9

10

-8

10

-7

10

-6

10

-5

10

-11

10

-10

10

-9

10

-8

10

-7

10

-6

10

-5

10

Concentración de péptido (M) 50

EBNA 3B EBNA 3BA2 EBNA 3BQ2 SIN PÉPTIDO

40

LY c29

30 20 10 0 -11

10

-10

10

-9

10

-8

10

-7

10

-6

10

-5

10

Concentración de péptido (M)

PÉPTIDO

SECUENCIA B*2702

EBNA3B

RRARSLSAERY

20

EBNA3BA2

-A---------

100

EBNA3BQ2

-Q---------

20

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RESULTADOS

IV.3. MODULACIÓN DE LA ESPECIFICIDAD EN LAS POSICIONES DE ANCLAJE P1, P3 Y PΩ, POR EL POLIMORFISMO DE HLA-B27. (Anexo 5) IV.3.1. Especificidad de B*2705, B*2704 y B*2706 por los residuos en P1, P3 y P9. Para determinar la contribución de las posiciones de anclaje secundario P1 y P3 y del anclaje primario P9, a la unión de péptidos a HLA-B27, se utilizaron tres series de nonámeros de poli-Alanina en los que conservando el anclaje principal Arg2, cada una de las tres posiciones fue sustituida por diferentes aminoácidos. La unión de cada análogo (EC50), se cuantificó con base a la unión del péptido control RRYQKSTEL, un ligando natural de B*2705, B*2704 y B*2706. La contribución relativa de cada residuo, se calculó como la unión relativa del análogo correspondiente respecto al péptido ARAAAAAAA (ARA7) (Figs. 8A, 8B y 8C). Este péptido, se unió con más afinidad a B*2704 y B*2706 (10 y 9 µM respectivamente) que a B*2705 (30 µM) lo que significa que el esqueleto de poli-Alanina interacciona más fuertemente en los dos primeros. Residuo P1: El residuo mas favorecido en B*2705 fue R (EC50 de RRA7= 9 µM), cuya contribución al anclaje respecto a Ala1 fue aproximadamente 3. K, H, G, I, M y los residuos aromáticos fueron aproximadamente equivalentes a Ala y los residuos ácidos, polares, y L y V redujeron claramente la afinidad. En general, la especificidad de B*2704 por residuos en P1 fue similar a B*2705 en lo referente al efecto negativo de los residuos ácidos, polares y alifáticos. Una diferencia fue la mayor preferencia de este subtipo por G (EC50 de GRA7= 6 µM) que por R. B*2706 se diferenció claramente de B*2704, en que sólo D estaba muy desfavorecido. La contribución a la unión de los residuos básicos R, K, H, los polares, Q, G y Y fue mayor en B*2706 que en los otros dos subtipos. Estos resultados indican que B*2705, B*2704 y B*2706 a pesar de tener una misma subcavidad A, muestran diferencias en sus preferencias por el residuo P1. Residuo P3: Los residuos en esta posición mostraron un amplio rango de afinidades de unión a B*2705. El más favorecido fue W (EC50 =5µM), y en general se apreció una preferencia por los residuos apolares. H, N, y Y fueron equivalentes a Ala y los residuos ácidos, T, Q, G y P estaban desfavorecidos.

RESULTADOS

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B*2704 se asemejó a B*2705 en general. Las principales diferencias con B*2705 fueron que Ala no era peor que otros residuos alifáticos de mayor tamaño y que N era peor que Ala en B*2704, pero no en B*2705. La especificidad de B*2706 por los residuos en P3 mostró importantes diferencias con B*2704. Así, H, R, y los residuos polares estaban especialmente favorecidos respecto a Ala en B*2706, y G, P y los residuos ácidos estaban menos desfavorecidos. Además, los residuos apolares alifáticos y los aromáticos eran más adecuados que Ala, pero Y estaba menos favorecida que muchos otros residuos, entre los que se incluían los aromáticos. Ala por si misma estaba entre los mejores residuos de B*2704, pero era peor que muchos otros en B*2706. Estos resultados indican que B*2704 y B*2706 difieren entre si, y también de B*2705, en las preferencias por el residuo P3. Residuo P9: El número de aminoácidos que se estudió en esta posición se limitó a los residuos básicos y apolares, por ser este el tipo de residuos C-terminales mayoritariamente presentado por B*2705. Adicionalmente se incluyó Pro, un residuo C-terminal presente entre los péptidos de HLA-B73, que muestra la misma preferencia que HLA-B27 por Arg2. Como era esperado, tanto los residuos básicos como los apolares se unieron en general bien a B*2705, pero no de forma equivalente. Entre los residuos aromáticos Y estaba favorecido, pero no F ni W. Asimismo, P estaba desfavorecida. En B*2704, F, W y P también estaban desfavorecidos, además de los residuos básicos y Y. Por último, en B*2706, y al igual que en B*2704, ni los residuos básicos, ni Y, W o P eran adecuados, pero B*2706 mostró una mayor preferencia que B*2704 por los residuos alifáticos y F. Estos resultados indican que una de las principales diferencias entre la especificidad de B*2704 y B*2706 por los residuos C-terminales, radica en la mejor aceptación de F por B*2706. IV.3.2. La unión de un péptido es el resultado de la contribución aditiva de varios residuos de anclaje. Para determinar si la afinidad de unión de un péptido es simplemente la suma de las contribuciones individuales de cada uno de los residuos de anclaje, o está influida por efectos cooperativos más complejos, se analizó la unión de dos ligandos naturales RRYQKSTEL y KRYKSIVKY y de distintos análogos de poli-Alanina en los que se conservaron una o varias de

las posiciones P1, P3, P7 y P9. Para calcular el EC50 de todos los análogos se utilizó como

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RESULTADOS

referencia el péptido RRYQKSTEL Primeramente, se midió en qué medida la introducción de esas posiciones de anclaje incrementaba o hacía decrecer la unión respecto a ARA7. Después se calculó la relación entre la unión relativa de cada ligando, o análogo portador de multiples posiciones de anclaje del ligando natural, y la suma de las uniones relativas de cada análogo de poli-Alanina, con uno sólo de los residuos de anclaje. Si la contribución de los residuos individuales es aditiva, esta relación debe ser 1. Si existen efectos interactivos entre los residuos de anclaje, esta relación se desviará de forma significativa de ese valor. Para tener en cuenta el error experimental, los valores de esa relación entre 1,5 (1,5:1) y 0,67 (1:1,5) se consideró que implicaban contribución aditiva. Como se aprecia en la (Figura 8D_a), el EC50 de la mayor parte de los péptidos portadores de varios anclajes del péptido RRYQKSTEL, (P1, P3, P7, P9) es resultado, en cuatro de cinco casos, del valor aditivo de sus correspondientes análogos portadores de un simple residuo. De manera similar el EC50 de los análogos con múltiples posiciones de anclaje del péptido KRYKSIVKY es, en cuatro de cinco casos, el resultado de la contribución de los análogos con

uno o pocos residuos de anclaje (Figura 8D_b), con un único caso (KRAAAAAAY, relación 0,6) que mostró una ligera desviación del rango 0,67-1,5. Estos resultados indican que en general, la eficiencia de unión de un péptido es simplemente una función aditiva de la contribución de los anclajes individuales; no obstante los efectos interactivos mútuos de las cadenas laterales pueden en ocasiones afectar a la unión. El papel que juegan las posiciones P4, P5, P6 y P8, se determinó a partir de la relacion EC50(ligando natural)/EC50(análogoP1-P2-P3-P4-P9) que mostró unos valores de 10/15=0,67 y 7,5/4,3=1,7 para RRYQKSTEL y KRYKSIVKY respectivamente. Este resultado muestra que su contribución a la unión es variable y dependiente del péptido.

RESULTADOS

-50-

Leyendas para las figuras 8A,8B,8C y 8D. (Ver página siguiente). Figuras 8A, 8B y 8C.

Figura 8D.

Unión relativa de diferentes análogos de poliAlanina a B*2705, B*2704 y B*2706.:

Relación entre la unión de los péptidos (a) RRYQKSTEL y (b) KRYKSIVKY. con distintos análogos en las posiciones de anclaje:

Cada análogo aparece representado en el eje de abscisas por el código de una letra del residuo introducido en la secuencia del péptido ARA7 en posición P1, P3 o P9. El péptido de referencia RRYQKSTEL (EC50: 8A=3 µM; 8B=4 µM; 8C=0,3 µM ) está marcado con un asterisco (*). La unión relativa de cada análogo (eje de ordenadas, en escala logarítmica) indica la relación entre el EC50 de ARA7 (8A=30 µM; 8B=10 µM; 8C=9 µM) y el del análogo correspondiente. Debido a que la concentración máxima que se utilizó de péptido fue 100 µM, la unión relativa menor que 0.3, que indica carencia de unión en este ensayo (EC50>100 µM), no puede medirse. En la gráfica se le asignó un valor de 0,25 por motivos de representación.

Las barras negras indican la unión relativa respecto al análogo ARA7, expresada como la relación molar entre el EC50 de ARA7 y el de cada péptido (abscisas). En los residuos cuyo efecto fue detrimental respecto a Ala (T7, K1), el descenso de la unión relativa se expresó como: 1-unión relativa. Las barras blancas indican el valor aditivo de las uniones relativas de los análogos de poli-Ala portadoras de sustituciones sencillas en P1, P3, P7 o P9. El efecto de V7 no se analizó de forma separada; en su lugar se utilizó el análogo ARAAAAVAY. Teniendo en cuenta el error experimental en la determinación de los valores de EC50, los valores representados por las barras negras y blancas se consideraron iguales, cuando su relación se encontraba entre 0.67 (1:1.5) y 1.5 (1.5:1). Los valores que excedieron esos margenes aparecen marcados con un asterísco (*).

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RESULTADOS

B*2705

A

B

B*2704 10

10

P1 1

1

0,1

0,1 K H R D E S T N Q G A V I L M F Y W 10

*

K H R D E S T N Q G A V I L M F Y W

*

H R D E S T N Q G A V I L M F Y W P

*

10

P3 1

1

0,1

0,1 H R D E S T N Q G A V I L M F Y W P

10

*

10

P9 1

1

0,1

0,1 K

R

A V I L M F Y W P

B*2706

K

*

R

A V I L M F Y W P

D

C

100

P1

10

RRYQKSTEL RRAAAAAAA ARYAAAAAA ARAAAATAA ARAAAAAAL

1

a 0

ARYAAATAA 0,1 K H R D E S T N Q G A V I L M F Y W 100

1

H R D E S T N Q G A V I L M F Y W P

*

P9

KRAAAAAAY

KRYAAAVAY K

R

A V I L M F Y W P

*

14

16

b 2

4

KRYAAAAAA

ARYAAAVAY

0,1

12

RRYAAATAL

ARYAAAAAY 1

10

ARYAAATAL

0

10

8

KRYKSIVKY KRAAAAAAA ARYAAAAAA ARAAAAAAY ARAAAAVAY

0,1 100

6

ARAAAATAL

P3

10

4

*

ARYAAAAAL

*

2

*

6

8

10

12

14

16

*

RESULTADOS

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IV.3.3. Distribución de los residuos P1, P3 y PΩ entre los ligandos naturales de B*2705. Conocida la contribución al anclaje de cada residuo, se procedió al estudio comparativo de su distribución entre 54 ligandos naturales de B*2705 (Tabla 10), con el propósito de determinar las reglas que rigen el uso de residuos en las posiciones de anclaje, por el repertorio peptídico natural de este subtipo. En función de la eficacia de unión de cada uno de los residuos, respecto al péptido de referencia ARA7, se establecieron cinco niveles, siendo el nivel 1 el de los residuos más favorecidos. (Tabla 9). Rango

Nivel

<10 µΜ 11-20 µΜ 21-40 µΜ 41-80 µΜ >80 µΜ

1 2 3 (ARA7) 4 5

P1

P3

PΩ

44% ( 24/54 )

85% ( 45/53 )

86% ( 44/51 ) -

48% ( 26/54 )

15% ( 8/53 )

14% ( 7/51 ) -

Tabla 9: Distribución porcentual de los residuos P1, P3 y P9 entre los ligandos naturales de B*2705.

Los resultados indican que las posiciones P3 y P9, utilizan residuos favorables (niveles 1 y 2) en el 85% de los casos, pero pueden acomodar también residuos desfavorables. La posición P1, es más permisiva y acomoda residuos desfavorables (niveles 4 y 5) en el 48% de los casos. Adicionalmente se siguen las siguientes normas de uso: Entre los, nonámeros y decámeros con PΩ subóptimo (niveles 3 a 5) (cinco nonámeros y dos decámeros) todos tienen un anclaje óptimo en P3 (nivel 1), independientemente del residuo presente en P1. De igual modo, todos los péptidos de estos tamaños, con un anclaje subóptimo en P3 (cinco nonámeros y tres decámeros) tienen un PΩ bueno (nivel 1), independientemente del residuo presente en P1. Este resultado indica que la presencia de un anclaje subóptimo en P3 o en P9, siempre se compensa con un buen anclaje en la otra posicion. Dieciocho de los 30 péptidos con un mal residuo de anclaje en P1 (niveles de 3 o >3), tienen tanto en P3 como en P9 un buen residuo de anclaje (niveles 1 y 2) y todos, como mínimo, un buen anclaje al menos en una de estas dos posiciones. Siete de los péptidos, que muestran un mal anclaje en P1 y P3 o P1 y P9 tienen en el residuo restante uno de nivel 1. Por lo tanto, un mal residuo de anclaje en P1 requiere un residuo óptimo al menos en una de las otras dos posiciones. Entre los péptidos de tamaño no-canónico (octámeros, undecámeros y dodecámeros), las

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RESULTADOS

tres posiciones estudiadas mostraron buenos anclajes (niveles 1 y 2), lo que sugiere que este tipo de ligandos, es más restrictivo en el tipo de residuo que puede ocupar sus posiciones P1, P3 y PΩ, aunque el número de ligandos conocidos de estos tamaños es aún limitado. Estas reglas, estudiadas en B*2705 tambien se cumplen con los escasos ligandos conocidos de B*2704 y B*2706 (Anexo 5, Tabla 2).

RESULTADOS

Secuencia

-54-

Valoración del residuo

Referencia.

Secuencia

Octámeros RRFFPYYV RRFTRPEH

Referencia.

4-1------1 5-1------1 1-5------1 4-1------1 1-2------1 4-1------1 4-X------1 4-2------1 1-5------1 5-5------1 4-1------1 1-1------4 4-1------4 1-2------X

-Este estudio-Este estudioHIV (*) [7] [6] [6] [6] [6] [6] -Este estudioHIVd (*) -Este estudio[6] [3]

1-2-------1 1-1-------1 2-1-------1

[7] [3] -Este estudio-

1-1--------1

[6]

Decámeros 1-1----1 1-1----X

[1] [2]

3-1-----1 1-2-----1 4-4-----1 1-1-----1 4-2-----1 1-2-----1 1-2-----1 4-1-----1 4-4-----1 5-1-----1 1-3-----1 3-2-----1 4-1-----1 3-3-----1 4-2-----1 1-1-----1 4-2-----1 1-1-----1 1-2-----1 1-2-----1 1-2-----1 1-2-----1 3-1-----1 4-2-----1 4-1-----1 4-3-----1 4-1-----1 1-1-----3 4-1-----4 4-1-----4 1-1-----4 4-1-----4 5-2-----X 4-2-----X

-Este estudiob [2] -Este estudio[3] -Este estudio[4] (*) [5] (*) [6] [6] [6] [2] [2] [2] [6] [2] (*) [3] -Este estudio[1, 7] [6] [2, 3]c [2] [2] [2,3] [2] [6] HIV (*) HIV (*) [2] [3, 7] -Este estudio[3] [3] [3] [8] (*)

KRFEETGQEL NRFAGFGIGL RRQDILDLWI GRFNGQFKTY RRYDRKQSGY GRWPGSSLYY GRKTGQAPGY GRILSGVVTK RKGGNNKLIK LRDNIQGITK KRWIILGLNK RRFVNVVPTF KRWQAIYKQF RRIKEIVKKH

Nonámeros ARLQTALLV RRYQKSTEL SRTPYHVNL RRLPIFSRL GRHGVFLEL RRIYDLIEL RRYPDAVYL GRFGSGMNM GRTFIQPNM LRFQSSAVM RRSKEITVR FRYNGLIHR KRFEGLTQR HRAQVIYTR SRYWAIRTR RRFMPYYVY SRVKLILEY RRFFPYYVY RRVLVQVSY RRISGVDRY RRIKEIVKK RRVKEVVKK ARLFGIRAK GRIDKPILK GRFEGTSTK GRAFVTIGK IRLRPGGKK RRWLPAGDA GRLTKHTKF KRFKEANNF RRFGDKLNF KRFSFKKSF TRYPILAGH KRVVINKDT

Valoración del residuo

Undecámeros RRYLENGKETL RRMGPPVGGHR WRLGSSDILNY

Dodecámeros RRFVNVVPTFGK (b)

Descrito previamente como octámero (ARLQTALL) por secuenciación de Edman [Rötzschke et al., 1994]. También encontrado como nonámero en B*2709 (ARLQTALLV) [Fiorillo et al., 1997].

(c)

Descrito también como nonámero en B*2701 [García et al., 1997c] y como decámero (RRISGVDRYY) en B*2703 [Boisgérault et al., 1996 ] y B*2710 [García et al., 1998].

(d)

Existe también una variante natural de este péptido con el cambio L6M.

[1] Paradela et al., 1998. [2] Jardetzky et al., 1991. [3] Rötzschke et al., 1994. [4] Brooks et al., 1993. [5] van Binnendijk et al., 1993. [6] Fiorillo et al., 1997. [7] Boisgérault et al., 1996. [8] Ugrinovic et al., 1997.

Tabla 10: Ligandos naturales de B*2705 y valoración de los residuos P1, P3 y PΩ. (a)

a

Los péptidos marcados con un asterisco son de origen bacteriano o viral (*). El resto pertenecen a proteínas endógenas celulares. En negrita se indican los valores asignados a cada residuo. X: valoración no analizada. Los péptidos derivados del HIV se obtyuvieron del HIV Molecular Immunology Database of Los Angeles National Laboratory. (http://hiv-web.lanl.gov/).

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RESULTADOS

IV.4. UNIÓN DE ANÁLOGOS NO PEPTÍDICOS A HLA-B27. (Anexo 7) Como una primera aproximación al posible uso de ligandos no-peptídicos como antagonistas en la respuesta inmune mediada por CTLs, en este estudio se analizaron las propiedades de unión de varios de estos ligandos a HLA-B27. Para ello se alteró la

OH

H2N

Aua: 11-Amino undecanoico.

región central, que comprende los residuos P4P8, en varios ligandos naturales de B*2705

O

(Tabla 11). Se pretendía con ello, conservar

O

O O

O O HB3

las propiedades de unión del péptido natural a O

la molécula de clase I, y simultaneamente alterar su región central, reconocida por el

O

O O

O O

O O HB4

TCR. En los análogos no peptídicos utilizados, la parte central fue sustituida por espaciadores

Figura 9: Estructura química de los tres espaciadores no peptídicos utilizados en los análogos. Aua: ácido 11-amino undecanoico. HB: (R)-3-Hidroxibutirato.

no peptídicos de diferente naturaleza química y longitud (Figura 9). El diseño de estos ligandos se realizó por técnicas de simulación de dinámica molecular, y su unión a HLA-B27 se analizó mediante un ensayo de estabilización de péptidos in vitro. Asimismo, la eficiencia de unión a B*2705, medida por este ensayo, se compararó con el parámetro de desnaturalización térmica (Tm) obtenido mediante dicroísmo circular. IV.4.1. Reemplazamiento de la parte central de epítopos naturales con un espaciador monofuncional. El ácido 11-amino undecanoico (Aua) es un espaciador monofuncional, que únicamente enlaza los residuos P3 y P9, pero no establece interacciónes con la molécula de clase I por carecer de cadenas laterales. En los cuatro análogos analizados con este espaciador, se apreció una pérdida general de afinidad respecto al péptido natural, tanto en el ensayo de unión in vitro como en su estabilidad térmica. Sin embargo las diferencias observadas por este último criterio fueron más acusadas (Tabla 11). La unión de los análogos de -Aua- a B*2704, fue muy ineficiente cuando el residuo P9 era básico, pero apenas se alteró cuando dicho residuo era apolar. Este resultado indica la importancia de un buen anclaje en P9 para la unión del análogo con este tipo de espaciador, y el

RESULTADOS

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papel decisivo de las interacciones secundarias entre el péptido y B*2704, cuando P9 es un mal anclaje para este subtipo. IV.4.2. Reemplazamiento de la parte central de epítopos naturales con espaciadores bifuncionales. En base a los resultados anteriores, se ensayaron dos tipos de espaciadores bifuncionales formados por oligómeros de (R)-3-hidroxibutirato (HB), uno trimérico -HB3- y otro tetramérico -HB4. Estos espaciadores, además de enlazar P3 y P9 forman polímeros químicamente estables, y pueden adoptar plegamientos conformacionalmente similares a los de los péptidos en estado libre (Plattner et al., 1993). Adicionalmente, poseen grupos metilo capaces de interaccionar en las subcavidades del sitio de unión del péptido. Con estos espaciadores se construyeron los correspondientes análogos del ligando de HLA-B27 QRLKEAAEK, y una variante con Ala1, ARLKEAAEK, con objeto de paliar posibles problemas en el ensayo de unión derivados de la

eventual ciclación de la Gln N-terminal. Ambos análogos mostraron eficiencias de unión in vitro muy diferentes (Tabla 11). El análogo (12) con -HB4- se unió 15 veces mejor que el (11) con -HB3-. La presencia del espaciador -HB4- siempre mejoró la unión respecto al péptido natural (7). Sin embargo, los valores de Tm que mostraron los análogos estudiados fueron similares (Péptidos 11 y 12, Tabla 11). Estos resultados indican que la introducción de grupos -HB4- en la parte central del espaciador, mejora la unión a B*2705 considerablemente, pero no afecta de forma significativa a su estabilidad térmica. IV.4.3. Modelado molecular de la unión de análogos con espaciadores no peptídicos a B*2705. Mediante la modelización, se predicen diferencias claras entre el péptido natural QRLKEAAEK y los análogos con espaciador no peptídico (Anexo 7, Figuras 2 y 5). En primer

lugar, el número de contactos establecidos por el análogo con -Aua- era menor, lo que explica la menor afinidad de los análogos con este espaciador respecto al péptido natural. La menor longitud del análogo con -HB3- impide la interaccion óptima con las subcavidades A y F, lo que no sucede con el análogo con -HB4- cuya mayor longitud le permitía incrementar el número de interacciones en ambos extremos, que son suplementadas por los contactos adicionales de los grupos metilo. Por tanto, la modelización teórica de las interacciones con HLA-B27 confirmó, al

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RESULTADOS

menos cualitativamente, las diferencias experimentales y proporcionó una posible interpretación molecular de estos resultados.

Péptido

EC50 (a)

Secuencia

Tm (ºC)(b)

P1-P2-P3

-Espaciador-

P9

B*2705

B*2704

1(c) 2

RRR RRR

WRRLT -Aua-

V V

1,2 4

0,8 1

52,8 ± 0,7 42,9 ± 0,3

3(d) 4

SRY SRY

WAIRT -Aua-

R R

3 8,6

1,4 100

46,3 ± 0,5 39,5 ± 0,2

5(e) 6

GRA GRA

FVTIG -Aua-

K K

1,8 7

6,4 >100

61,9 ± 0,3 48,1 ± 0,4

7(f) 8 9 10

QRL QRL QRL QRL

KEAAE -Aua-HB3-HB4-

K K K K

10 40 2,5

11 12

ARL ARL

-HB3-HB4-

K K

20 1,6

62,8 ± 0,7 46,5 ± 0,2

63,2 ± 0,6 62,1 ± 0,7

Tabla 11: Valores de EC50 y Tm de los péptidos naturales y sus análogos no peptídicos. (a) EC50: Concentración de ligando a la cuál la fluorescencia de HLA-B27 en las células RMA-S, es la mitad de la fluorescencia máxima obtenida con el péptido natural. (b) Tm: Punto medio del valor térmico de desnaturalización del heterodímero formado por la cadena pesada de B*2705, la β2m y el ligando.

(c) Proteína latente de membrana del EBV (236-244), (Brooks et al., 1993). (d) Nucleoproteína del virus Influenza A (383-391), (Huet et al., 1990). (e) Glicoproteína 120 del virus del SIDA (314-322), (Jardetzky et al., 1991). (f) Proteína DnaK de E. coli (260-268), (Rognan et al., 1995).

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V. DISCUSIÓN

DISCUSIÓN

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V. DISCUSIÓN V.1. POLIMORFISMO DE HLA-B27 Y SOLAPAMIENTO DE REPERTORIOS PEPTÍDICOS ENTRE SUBTIPOS.

V.1.1. Influencia del polimorfismo de HLA-B27 sobre la especificidad por el residuo Cterminal del péptido. Diferencias entre B*2704 y B*2706.

L

a teoría del péptido artritogénico implica que los subtipos asociados a enfermedad, deben de ser capaces de presentar un mismo péptido unido selectivamente a los subtipos asociados a enfermedad. Por esta razón, es particularmente importante

conocer el grado de solapamiento de los repertorios peptídicos unidos a los diferentes subtipos de HLA-B27, y las diferencias entre los repertorios de los subtipos asociados diferencialmente a enfermedad. Contrariamente a lo que ocurre en la subcavidad B, cuya estructura es común entre los subtipos de HLA-B27 y está optimizada para unir péptidos con Arg2, la mayor parte del polimorfismo de esta molécula se concentra en las subcavidades C y F, lo que se traduce en distintas especificidades por el residuo C-terminal. B*2705 une péptidos con residuos C-terminales básicos, alifáticos o aromáticos. Muchos de los péptidos presentados por B*2702 se unen in vitro y probablemente también in vivo a B*2705 (García et al., 1997b). Este solapamiento de repertorios peptídicos entre ambos subtipos concuerda cualitativamentecon los patrones de reactividad cruzada de CTLs alorreactivos (López et al., 1994). Por tanto B*2705 y B*2702 son un ejemplo de dos subtipos asociados a EA, con un grado importante de solapamiento de sus repertorios peptídicos. Puesto que B*2702 no une péptidos con residuos C-terminales básicos, éstos probablemente no están implicados en la patogénesis de la EA. A pesar de las similitudes estructurales entre B*2704 y B*2706, sólo el primero está asociado a enfermedad (López-Larrea et al., 1995), y sus especificidades por el residuo Cterminal muestran marcadas diferencias. B*2704 une preferentemente in vitro, péptidos con residuos C-terminales alifáticos y aromáticos, similares a los presentados por B*2702 in vivo. Algunos péptidos con residuos Cterminales básicos también se unen a B*2704 in vitro, sin embargo tales péptidos no se han

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detectado entre los ligandos naturales de este subtipo. B*2706 por su parte, discrimina en mayor medida que B*2704 entre los residuos Cterminales polares y apolares, lo que se concreta en una mayor preferencia que B*2704 por Leu y Phe y menor por residuos básicos y Tyr. Ello sugiere que el repertorio de péptidos que B*2706 comparte con otros subtipos, incluye principalmente a los péptidos con Leu y Phe C-terminales. Las diferencias de especificidad observadas in vitro entre B*2704 y B*2706 se correlacionan con las que muestran in vivo, como se deriva de la secuenciación de los pooles de péptidos eluídos de ambos subtipos (García et al., 1997b). El aumento de la preferencia por residuos C-terminales apolares tras la eliminación de los dos residuos ácidos Asp77 y Asp116, explica las especificidades C-terminales de B*2704 y B*2706. No obstante, la ausencia de uno sólo de estos residuos en B*2704, no es suficiente para impedir totalmente la unión de péptidos con residuos C-terminales básicos. La preferencia que muestra B*2706 por Leu y Phe, y la unión ineficiente de Tyr Cterminal, es probablemente debida al cambio D→Y116. El papel de esta posición ha sido analizada en dos estudios previos en los que se analizaban mutaciones distintas, pero en ambos tanto Leu como Phe eran los dos residuos C-terminales más adecuados, cuando D116 era mutado. El cambio de Asp por Phe disminuía la aceptación de residuos C-terminales básicos (Parker et al., 1994). Esta especificidad se explicaría por la incapacidad de Phe para formar puentes de hidrógeno. En el otro estudio (Fiorillo et al., 1995), B*2709 cuyo residuo 116 es His, era incapaz de unir péptidos de poli-Alanina con Arg o Tyr C-terminal pero sí podía unir Lys. Puesto que B*2709 tampoco se asocia a EA, este estudio junto con los de B*2706 sugiere que el residuo tiene un papel clave en determinar la susceptibilidad a EA. La introducción de residuos ácidos en las posiciones 114 y 152, no altera la especificidad de la subcavidad C/F debido a su localización, pero afectan notablemente a la unión in vitro de los péptidos estudiados. Estos resultados sugieren que es posible alterar la especificidad por péptidos unidos sin alterar las preferencias por los anclajes primarios, debido probablemente a la modulación de las interacciones con residuos de anclaje secundarios (por ejemplo P7). En el caso concreto de E152 (B*2710), este subtipo es muy distinto antigénicamente de B*2705, puesto que es el que presenta una menor reactividad cruzada (Calvo et al., 1990). Sin embargo, el análisis del repertorio peptídico natural de B*2710 reveló una sorprendente homología con el de HLA-B27 (García et al., 1998 / Anexo 8). Por tanto el efecto de la mutación E152 es mucho mayor sobre el reconocimiento por los TCR que sobre la especificidad de unión

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de péptidos. Estudios de modelización sugieren que ésto es debido a que el residuo 152 en HLAB27 es accesible al TCR (García et al., 1998 / Anexo 8; Figura 5). Cuando se compara la unión de muchos de los péptidos a B*2704, S77 y E152, se observa que la mutación S77 compensa el efecto negativo del residuo E152. De igual modo, las afinidades de unión similares a Y116 y al doble mutante D114Y116 muestran una compensación de la mutación Y116 sobre la mutación D114. Estos efectos compensatorios que modulan la unión de péptidos, se correlacionan con resultados previos en los que se observaron efectos compensatorios de las mutaciones en el reconocimiento por CTLs alorreactivos (López et al., 1992; Villadangos et al., 1994). Estos efectos compensadores, podrían suponer una ventaja evolutiva en el polimorfismo de HLA-B27. Debido a que dos mutaciones generadas mediante conversión génica, un mecanismo evolutivo frecuente en este sistema (López de Castro, 1989; Parham et al., 1995), tienen un efecto menos disruptivo que una mutación puntual. La ausencia de Asp116, y la menor capacidad para unir péptidos con C-terminal Tyr, son características que comparten B*2706 y B*2709 y que diferencian a estos subtipos de otros asociados a enfermedad: B*2705, B*2702 y B*2704. Esta correlación podría sugerir que un posible péptido artritogénico debería tener Tyr lo que restringiría en gran medida el número de candidatos. B*2707, aunque tampoco presenta el motivo Tyr C-terminal (Tieng et al., 1997) sí está asociado a enfermedad. Sin embargo, es posible que este subtipo pudiera presentar péptidos con Tyr C-terminal en niveles no detectables por secuenciación de Edman. V.1.2. El polimorfismo en la cavidad C/F determina parcialmente la especificidad de la subcavidad B. Los resultados derivados del estudio realizado con B*2701, B*2702 y los mutantes que mimetizan sus cambios, todos localizados en la cavidad C/F, pusieron de manifiesto la preferencia de B*2701 por péptidos con residuo Leu, Tyr y Phe C-terminal de forma similar a B*2702 (Rötzschke et al., 1994). Estas preferencias se explican por la mayor hidrofobicidad de la cavidad C/F de ambos subtipos, respecto a B*2705. Adicionalmente, muchos de los péptidos secuenciados de B*2701 son compartidos por otros subtipos asociados a enfermedad, lo que es compatible con una posible asociación de B*2701 a EA. De hecho uno de los pocos individuos tipados como B*2701+, padecía esta enfermedad. La principal diferencia entre B*2701 y B*2702 reside en las

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preferencias por el residuo en P2. Este residuo, que está conservado entre los subtipos de HLAB27, es Arg2; la Gln2 aunque compatible con la unión de péptidos in vitro a varios subtipos, es un residuo sobóptimo (García et al., 1997a; Villadangos et al., 1995; Galocha et al., 1996 / Anexo 1; Parker et al., 1994; Fukazawa et al., 1994; Raghavan et al., 1996). Sin embargo, B*2701 es capaz de unir in vitro y presentar in vivo péptidos tanto con Gln2 como con Arg2 con eficiencia similar. Esta característica singular de B*2701 radica en el cambio D→Y74, como se deduce de la presencia de Gln2 y Arg2 entre los péptidos que unen in vivo tanto B*2701 como en el mutante Y74 (Anexo 2). El efecto que ejerce la mutación Y74, situada en la subcavidad C/F, sobre la especificidad de la subcavidad B, tiene su explicación en el residuo Lys70. Este residuo, casi exclusivo de HLA-B27, se localiza próximo a la subcavidad B, pero no interviene en las interacciones con el residuo P2. En B*2705, su cadena lateral se orienta hacia el exterior de la subcavidad B (Madden et al, 1992) estableciendo un puente salino con el residuo Asp74. En B*2701, la mutación Y74 impide la formación de ese enlace permitiendo la reorientación de la cadena lateral de la Lys70 hacia la subcavidad B, donde puede interaccionar con residuos peptídicos de Gln2. Estudios recientes realizados en nuestro laboratorio (Krebs et al. 1999 / en revisión), han confirmado experimentalmente el papel crítico de la Lys70 en la especificidad de B*2701 por Gln2. En estos estudios, la mutación de Lys70 a Ala70 en B*2701, revierte la especificidad a Arg2 exclusivamente. Adicionalmente, los estudios de modelización molecular sugieren que la especificidad dual de B*2701 por Arg2 y Gln2 se debe a la capacidad de Lys70, en este subtipo, para adoptar dos estados rotaméricos distintos dependiendo del residuo P2. Como se ha dicho, si P2 es Gln, Lys70 se orienta hacia la subcavidad B para interaccionar con Gln2; si P2 es Arg, Lys70 se reorienta en una conformación diferente hacia fuera de la subcavidad B, preservando el mismo modo de interacción de la Arg2 en esta subcavidad, que en B*2705. La mala unión general de muchos de los péptidos al mutante A81 se explica porque la ausencia de Leu81 impide las interacciones con los residuos C-terminales apolares o con la porción alifática de los básicos (Madden et al., 1992). Finalmente, la mejor unión de algunos péptidos a B*2701 o B*2702 que al mutante A81, sugiere la existencia de efectos compensatorios de los otros cambios. V.1.3. Análisis del efecto del polimorfismo de la subcavidad A sobre la unión de péptidos.

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HLA-B*2703 se diferencia de B*2705 en un solo aminoácido, Y59H, ubicado en la subcavidad A. Esta mutación es la responsable de que B*2703 presente un subconjunto de los péptidos presentados por B*2705 (López et al, 1994; Villadangos et al, 1994) lo que hace interesante el estudio del efecto que tiene esta mutación en la unión de péptidos. En la estructura cristalográfica de HLA-B*2705 (Madden et al, 1992), las cadenas laterales de los aminoácidos Tyr7 y Tyr171 establecen puentes de hidrógeno con el extremo N-terminal del péptido. Ambas cadenas interaccionan adicionalmente, por mediación de una molécula de agua, con las cadenas laterales de los residuos cercanos Tyr59, Glu45 y Tyr171. (Figura 6 / Anexo 3) En B*2703, se produce la distorsión de esa red de enlaces. La molécula de agua desaparece y el extremo N-terminal del péptido se une a la His59, en lugar de a Tyr7. Como consecuencia, se pierden el enlace directo con Tyr171 y cinco interacciones mediadas por la molécula de agua. La desaparición de la molécula de agua que enlaza la subcavidad A con Glu45 en la subcavidad B, probablemente refuerza la interacción de este residuo con Arg2. En el caso en que P2 es Gln, los enlaces en la subcavidad B de B*2705 se debilitan, principalmente con Thr24 y Glu45 y se generan nuevos reordenamientos conformacionales en las interacciones con P1. Gln2 es más tolerado en B*2705, debido a que Tyr59 aún puede unirse a Tyr7. Adicionalmente el enlace Cα−Ν del péptido unido, sufre una rotación que establece un nuevo puente de hidrógeno con Glu63 sin perder el enlace con Tyr7. La cadena lateral de Gln2 establece además puentes de hidrógeno con Glu45 y Tyr99 reforzando las interacciones en la subcavidad B. Estas interacciones no se producen en B*2703, como explica la baja afinidad de péptidos con Gln2 a este subtipo (Figuras 7 y 8 / Anexo 3). Tanto en B*2705 como en B*2703, la presencia de Gln2 induce una desestabilización del extremo C-terminal. Esta situación ya detectada en otros péptidos (Rognan et al., 1994), sugiere la posible función estabilizadora de la región N-terminal para el resto del péptido. En los péptidos analizados, el residuo de Arginina en P1 es accesible a la formación de dos puentes salinos con Glu63 y Glu163, sin embargo esto no supone una ventaja significativa sobre la unión in vitro del análogo con Alanina. La cadena lateral de Alanina puede interaccionar con residuos apolares conservados de la subcavidad A como (Met5 y Trp167), lo que explicaría la similar afinidad de unión de péptidos tales como RRYQKSTEL y ARYQKSTEL. No obstante, hay estudios que indican la preferencia de B*2703 por residuos básicos (Colbert et al., 1994) y la

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presencia mayoritaria de éstos entre los péptidos que une in vivo (Boisgérault et al., 1996; Griffin et al., 1997) que no pueden explicarse mediante este modelo, aunque es posible la formación de puentes salinos entre varios rotámeros de la cadena lateral básica de P1 y los residuos cargados negativamente cercanos a la subcavidad A. En conclusión, el efecto del cambio Y59→H presente en B*2703 tiene varios efectos simultáneos que consisten en: (i) una disrupción de la red de puentes de hidrógeno que se establecen en la subcavidad A, (ii) reordenamientos de las interacciones en la subcavidad B y (iii) debilitamiento general de las interacciones que se producen con el extremo C-terminal del péptido. Estos efectos pueden afectar en gran medida al conjunto de péptidos presentados por B*2703 y hacer la unión de éstos más dependiente de la presencia de residuos básicos en P1.

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V.2. RELACIÓN ENTRE LA UNIÓN DE PÉPTIDOS Y LA SELECCIÓN DE EPÍTOPOS POR CÉLULAS T.

Los resultados de este estudio muestran: (i) la moderada influencia del polimorfismo en la unión de péptidos de EBV restringidos por HLA-B27, (ii) la inexistencia de correlación entre la unión promiscua de los péptidos virales a varios subtipos y su antigenicidad y/o inmunogenicidad en el contexto de un subtipo particular, (iii) el motivo de anclaje Arg2, no es necesario para mantener la estructura antigénica del péptido. La moderada incidencia que muestra el polimorfismo entre los subtipos B*2701-B*2706, en la unión de péptidos de EBV, que poseen residuos C-terminales alifáticos o aromáticos, es consistente con el predominio de este tipo de residuos entre los ligandos naturales de los seis subtipos (García et al., 1997a; Jardetzky et al., 1991; Boisgérault et al., 1996; Rötzschke et al., 1994; García et al., 1997b). En general, no existe una correlación entre la unión a un subtipo y su inmunogenicidad en el contexto del mismo. Este es el caso del péptido EBNA3B, restringido por B*2702 y de LMP2, restringido por B*2704, que muestran una buena unión a todos los subtipos, en algunos casos mejor incluso que al subtipo por el que están restringidos. A pesar de que el desarrollo de una respuesta mediada por CTLs requiere habitualmente una alta afinidad al elemento de restricción (Sette et al., 1994), en el estudio presente no se aprecia una correlación directa entre la afinidad de unión a un subtipo y su inmunogenicidad en el contexto del mismo. La inmunogenicidad parece correlacionarse mejor con la estabilidad del complejo MHC-péptido (Van der Burg et al., 1996; Levitsky et al., 1996). En un trabajo reciente (Brooks et al., 1998), EBNA3B, cuya unión in vitro es tan buena a B*2705 como a su elemento de restricción B*2702, mostró una unión más estable a B*2702 que a B*2705 que se correlacionó con la ausencia de reconocimiento del péptido en el contexto de B*2705. Por lo tanto, las diferencias de estabilidad, no evidentes en el ensayo in vitro, pueden ser las responsables de la inmunogenicidad de un péptido en el contexto de un subtipo y no de otros. Aunque múltiples factores contribuyen a limitar el número de péptidos inmunogénicos en una respuesta antiviral, tales como la afinidad, la acción de los proteosomas, el transporte mediado por TAP, el repertorio de células T y la supresión de la respuesta de células T por otros péptidos inmunodominantes (Deng et al., 1997); en este estudio los tres péptidos virales se unían significativamente a todos los subtipos y además eran inmunogénicos, al menos en el contexto de un subtipo, reduciendo las posibles variables, exclusivamente al de la capacidad de los repertorios de células T para reconocer la estructura del complejo péptido-MHC.

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El hecho de que la estructura del complejo péptido-MHC depende del subtipo, es evidente en la ausencia de correlación entre la unión de un péptido a varios subtipos, y su reconocimiento en el contexto sólo de algunos. Así, los CTLs restringidos por B*2704 para el péptido LMP2 no reconocían al péptido en el contexto de B*2702 ni de B*2705, confirmando observaciones previas (Brooks et al., 1993); los CTLs restringidos por B*2705 o B*2702 no reconocían al péptido EBNA3C unido a B*2704; los CTLs restringidos por B*2705 o B*2702 no reconocían al péptido en el contexto de B*2704, y la mayoría de los efectores contra B*2705 no reaccionaban de forma cruzada con B*2702. El reconocimiento por CTLs activados de un péptido restringido por un subtipo, implica un cambio en la estructura del epítopo tras la unión del péptido a diferentes subtipos, bien debido a un cambio conformacional del propio epítopo, o bien a que el polimorfismo entre subtipos altera la estructura del complejo o la interacción con el TCR. Esta última posibilidad es probablemente la responsable de la ausencia de reactividad cruzada entre B*2704 y B*2705 o B*2702. La razón de esto es que B*2704 difiere de los otros dos subtipos en el cambio E152→V. Esta mutación que no parece afectar mucho a la presentación in vivo de los mismos péptidos naturales que B*2705, ni altera su conformación, tiene sin embargo efectos drásticos en el reconocimiento por el TCR (García et al., 1998 / Anexo 8). Por lo tanto, el polimorfismo de HLA-B27 modula el reconocimiento por células T mediante factores adicionales a la simple afinidad de unión de péptidos. Las diferencias mayores entre subtipos residen en su diferente modulacion de la inmunogenicidad y antigenicidad de los péptidos unidos, más que en su diferente especificidad de unión de péptidos. La modulación de la antigenicidad e inmunogenicidad de un péptido en el contexto de diferentes subtipos, tiene claras implicaciones en la patogenia de HLA-B27. La hipótesis que sugiere la unión selectiva de un péptido artritogénico a los subtipos asociados a enfermedad tiene ahora otra alternativa adicional, consistente en que dicho péptido artritogénico pueda unirse a varios subtipos, pero sólo ser relevante en el contexto de algunos. En este estudio, péptidos que carecían del motivo Arg2 podían ser reconocidos por CTLs restringidos por el péptido con Arg2, lo que implica que su estructura antigénica reconocida no era alterada tras eliminar un motivo de anclaje principal. Evidencias adicionales como la reacción cruzada de CTLs con péptidos carentes de motivos canónicos (Malarkannan et al., 1996), la secuenciación de péptidos carentes de Arg2 en B*2701 (García et al., 1997c / Anexo 2) y en B*2705 de ratas transgénicas (Simmons et al., 1997) y la demostración de que los péptidos

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con Gln2 se unen in vitro a muchos subtipos de HLA-B27, sugiere que estos podrían constituir una fracción menor del conjunto total de péptidos en subtipos diferentes a B*2701. Por tanto, la búsqueda de posibles péptidos artritogénicos, en particular autopéptidos, no debería basarse exclusivamente en los motivos canónicos. Péptidos con motivos no canónicos, debido a su baja afinidad y escasa presentación in vivo, podrían eludir la autotolerancia mejor que los péptidos con Arg2 y convertirse en dianas de CTLs autorreactivos generados tras una infección artritogénica.

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V.3. LA ESPECIFICIDAD PEPTÍDICA DE LOS SUBTIPOS DE HLA-B27 ES MODULADA EN MULTIPLES POSICIONES DE ANCLAJE.

La variabilidad de los residuos en P1, P3 y PΩ determina en gran medida las propiedades de unión del péptido, pero la contribución de cada una de las posiciones está jerarquizada según el orden P9>P3>P1. Adicionalmente, el hecho de que muchos de los residuos sean inapropiados en cualquiera de estas tres posiciones indica que la unión del péptido depende tanto de los efectos positivos como de los negativos. La exploración de los residuos presentes entre los ligandos naturales de B*2705 revela un predominio de los residuos más adecuados en las posiciones P3 y P9; no obstante, la presencia de un residuo subóptimo en una de estas posiciones es tolerada, siempre y cuando se compense con la presencia en la otra posición de anclaje de un residuo óptimo. En el análisis de la distribución de residuos entre los ligandos naturales de B*2705 no se aprecian excepciones a esta regla, sugiriendo que si se excluyen los factores implicados en el procesamiento y transporte, aún insuficientemente caracterizados (van Endert et al., 1995; Uebel et al., 1997; Daniel et al., 1998; Peh et al., 1998), la limitación del número de ligandos naturales se basa en este tipo de restricciones. El residuo P1 es el más permisivo de los tres, y su contribución a la unión es menor, pero esta permisividad está condicionada por la presencia de un buen anclaje en P3 o P9. Teniendo en cuenta que la contribución aditiva de las tres posiciones, junto con la posición P2 (Arg2), da cuenta de la mayor parte de la afinidad del péptido natural, la predicción de epítopos naturales puede reducirse al estudio de los residuos P1, P3 y P9 obviando el papel de los residuos de las posiciones centrales, cuya menor contribución permite su sustitución por espaciadores no peptídicos que no alteran en gran medida su afinidad (Rognan et al., 1995; Krebs et al., 1998 / Anexo 6; Poenaru et al., en prensa / Anexo 7). A pesar de que no se conocen muchos ligandos naturales de tamaños distintos a los canónicos, de nueve o diez residuos, la unión de ligandos con tamaños no canónicos sólo parece posible cuando presentan buenos anclajes en las tres posiciones P1, P3 y PΩ. Un aspecto relevante de la comparación entre B*2705 y B*2704 reside en las diferentes especificidades por PΩ. En concordancia con su ausencia in vivo (García et al., 1997b) y su mala unión in vitro (Tanigaki et al., 1994; Galocha et al., 1996 / Anexo 1), los residuos PΩ básicos no están favorecidos en B*2704. Adicionalmente, los residuos C-terminales Y y F aunque no están favorecidos, son presentados de forma natural por B*2704 (García et al., 1997b) probablemente debido a que la estabilización por los otras posiciones de anclaje es suficiente para permitir la unión de ligandos con estos residuos. Por lo tanto, el solapamiento de repertorios peptídicos de B*2705 y

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B*2704, probablemente consiste sobre todo en péptidos con residuos C-terminales alifáticos, y aquellos con Y y F que además poseen en P3 residuos apolares. Para entender la base molecular de su diferente asociación a enfermedad, es fundamental analizar las diferencias entre B*2704 y B*2706. Las diferencias de B*2706 con B*2704 se localizan en posiciones que pueden influir directamente en la interacción con los residuos P3 y P9 de los péptidos unidos. Los resultados obtenidos concuerdan plenamente con los estudios previos de secuenciación que muestran la mayor preferencia de B*2706 por residuos C-terminales alifáticos y F, así como la ausencia de Y en PΩ (García et al, 1997b). Además, en los estudios in vitro, los residuos alifáticos están más favorecidos que Y en B*2706, pero no en B*2704 (Galocha et al., 1996 / Anexo 1). La ausencia de Y se explicaría por el mejor ajuste en B*2706 de los residuos alifáticos voluminosos y F que de Y, de forma que los péptidos con Y competirían en desventaja para unirse in vivo a B*2706. B*2704 y B*2706, muestran además otras diferencias en la preferencias por P3 y P1. Lo más destacado es la distinta especificidad por el residuo P3, especialmente una mejor aceptación de R, N y Q por B*2706 y la peor de A respecto a otros residuos. Estas diferencias son probablemente debidas a la presencia del residuo D114 en lugar de H114. Las diferencias en el residuo P1, entre subtipos que comparten una subcavidad A común, sugiere la existencia de efectos a larga distancia de posiciones polimórficas alejadas de esta subcavidad. La diferente especificidad de B*2704 y B*2706 en múltiples posiciones de anclaje implica que su asociación diferencial a enfermedad puede no correlacionarse exclusivamente con la incapacidad de B*2706 para unir Tyr C-terminal, sino también por la modulación de la especificidad por los residuos en P1 y sobre todo en P3. Por lo tanto B*2704 y B*2706, aunque comparten ligandos comunes pueden diferir en los repertorios de péptidos que presentan y muchos péptidos con Arg2 y residuo C-terminal compatible con la unión tanto a B*2704 como a B*2706 pueden sin embargo unirse exclusivamente a un subtipo.

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V.4. UNIÓN DE ANÁLOGOS NO PEPTÍDICOS A HLA-B27. La supresión de la reactividad por CTLs, puede lograrse mediante la utilización de ligandos modificados que al reaccionar con el TCR induzcan anergia, un fenómeno llamado antagonismo. Debido a los numerosos detalles conocidos de las interacciones entre un péptido y la molécula del MHC, el reemplazamiento en un péptido antigénico de la parte central reconocida por el TCR, por espaciadores orgánicos no peptídicos que conserven los residuos de anclaje a la molécula de clase I, es posiblemente una buena estrategia para diseñar ligandos que funcionen como antagonistas de péptidos antigénicos. La ventaja de tales compuestos sobre los antagonistas peptídicos, para su utilización in vivo, reside en su alta resistencia a la acción de proteasas y sus mejores propiedades farmacocinéticas (Ishioka et al., 1994). La modificación de la porción central del péptido con espaciadores no peptídicos, debe mejorar en lo posible tanto su afinidad de unión como su estabilidad. En los análogos estudiados con espaciador de -Aua- se observa una disminución de la afinidad, respecto al péptido natural, dependiente del subtipo al que se une. Su naturaleza monofuncional, permite la unión covalente entre los residuos P3 y P9 pero no el establecimiento de contactos adicionales en las subcavidades centrales de la molécula de clase I. La ausencia de grupos funcionales hace a estos análogos muy dependientes del buen anclaje en P9 que queda claramente reflejada en los valores de baja afinidad que muestran los análogos con P9 básico unidos a B*2704. Los espaciadores bifuncionales formados por trímeros y tetrámeros de -HB-, además de servir de enlace entre P3 y P9, poseen grupos funcionales metilo capaces de establecer contactos adicionales en el surco de unión del péptido. Sin embargo, los contactos sólo son posibles de forma óptima cuando la longitud del espaciador es apropiada, permitiendo el contacto de ambos extremos del análogo con las subcavidades A y F; en este sentido el análogo con -HB3- no cumple este requisito, una característica que lo diferencia claramente del análogo con el espaciador de -HB4-, que si lo cumple. El incremento de afinidad medido in vitro, es debido al anclaje adicional que proporcionan los dos grupos metilo del espaciador -HB4-, éstos interaccionan con las subcavidades C y E. El mayor número de interacciones determina una unión de este análogo superior a la del ligando natural. En este sentido la utilización del estereoisómero (R) no es trivial, puesto que la conformación estereoisomérica (R o S) de los sustituyentes es importante para mejorar la afinidad (Poenaru et al., en prensa /Anexo 7).

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DISCUSIÓN

En este estudio se observaron discrepancias en los valores comparativos de afinidad entre ligandos de HLA-B27 y sus análogos no peptídicos dependiendo de que se midiera su unión in vitro (EC50) o su estabilidad térmica (Tm). Estas discrepancias se explican porque el ensayo de unión in vitro, está muy influído por la cinética de asociación del péptido a la molécula de MHC, mientras que la Tm es una medida de la estabilidad del complejo y se relaciona directamente con la cinética de disociación del péptido. ™ RESUMEN Y DISCUSIÓN GENERAL: Si el papel patogénico de HLA-B27 reside en su función presentadora de péptidos, el conocimiento de las pautas que determinan el solapamiento de repertorios peptídicos entre subtipos, así como el reconocimiento de dichos péptidos en función del subtipo al que están unidos, son cuestiones fundamentales. En esta tesis se han definido algunos aspectos importantes de la modulación del repertorio peptídico por el polimorfismo de HLA-B27. Se ha analizado la relación entre la unión de péptidos y su inmunogenicidad y antigenicidad, y se han estudiado algunos aspectos de la unión de ligandos no peptídicos a HLA-B27. El polimorfismo de HLA-B27 modula la unión de péptidos a tres niveles: En primer lugar, el polimorfismo de las posiciones localizadas en la subcavidad C/F afecta directamente a la especificidad por el residuo C-terminal, que es un anclaje principal a HLAB27. En consecuencia, limita o impide la aceptación de residuos C-terminales básicos en múltiples subtipos. Además introduce una importante diferencia entre B*2704 y B*2706, dos subtipos asociados diferencialmente a EA, en cuanto a que restringe la aceptación de Y Cterminal en este último subtipo. En segundo lugar, algunas posiciones polimórficas localizadas en una determinada subcavidad modulan la especifidad por residuos peptídcos que interaccionan en subcavidades distintas. En esta tesis se han analizado dos efectos contrapuestos. El poliformismo de B*2703, localizado en la subcavidad A, fortalece la interacción de Arg2 en la subcavidad B y afecta a la estabilización del extremo C-terminal del péptido. Por otra parte, la mutación D74Y en B*2701, localizada cerca de la subcavidad C/F, favorece la interacción de Gln2 en la subcavidad B por un efecto indirecto mediado por la Lys70.

DISCUSIÓN

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En tercer lugar, el polimorfismo de B27 modula la especifidad en posiciones secundarias de anclaje. Esto es particularmente relevante, ya que indica que la asociación diferencial de B*2704 y B*2706 a EA, no se correlaciona solamente con la aceptación de Y C-terminal, sino con una modulación más compleja que incluye, adicionalmente, otras posiciones de anclaje. Las interrelaciones entre los repertorios peptídicos de los dos subtipos se hacen más complejas por el hecho de que las diversas posiciones de anclaje pueden tolerar en mayor o menor medida residuos desfavorecidos, que son compesados por la presencia de buenos anclajes en otras posiciones. El estudio efectuado con péptidos virales pone de manifiesto que más allá de las diferencias y similitudes en la especifidad de unión de péptidos, las diferencias funcionales entre subtipos dependen de la modulación adicional que ejerce el polimorfismo sobre la inmunogenicidad y antigenicidad de los péptidos unidos. En este estudio se demuestra que no existe una correlación entre la eficiencia con la que un péptido se une HLA-B27, y su capacidad para estimular una respuesta inmune o de ser reconcido por CTLs activados. Estos datos están de acuerdo con los conceptos de que la estabilidad del complejo MHC-péptido es el determinante crítico de la inmunogenicidad, y con que un CTL activado puede reconocer un péptido unido con muy baja afinidad a MHC si la conformación del epítopo está conservada. Finalmente, se han explorado las propiedades de unión a HLA-B27 de ligandos no peptídicos en los que los residuos P4-P8 fueron sustituidos por varios espaciadores orgánicos. Se ha demostrado que estos ligandos se unen a HLA-B27 con una afinidades que pueden ser superiores a la del péptido natural, dependiendo de la naturaleza química del espaciador. Estos estudios abren una vía a un ulterior análisis sobre el posible uso de estos compuestos en la modulación de la respuesta citotóxica restringida por HLA-B27.

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VI. CONCLUSIONES

CONCLUSIONES

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VI. CONCLUSIONES ™ Los subtipos B*2704 y B*2706, asociados diferencialmente a enfermedad, difieren en la menor preferencia de B*2706 por Tyr C-terminal y en su especificidad por residuos de anclaje secundario, en particular en P3. ™ B*2701 es el único subtipo conocido de HLA-B27 que une de forma significativa péptidos con Gln2 in vivo. La mutación Y74 es la responsable de esta característica y su efecto es indirecto a través de la Lys70. ™ Por lo tanto un residuo polimórfico (D74Y) puede ejercer efectos sobre la especificidad por residuos del péptido que interaccionan en regiones alejadas (cavidad B). Estos efectos están mediados a través de un residuo conservado (Lys70). ™ El cambio Y59→H en B*2703 tiene varios efectos simultáneos (i) ruptura de la red de puentes de hidrógeno en la subcavidad A, (ii) reordenamiento de las interacciones en la subcavidad B y (iii) debilitamiento general de las interacciones con el extremo C-terminal del péptido. Estos efectos hacen a B*2703 más dependiente del anclaje en P1. ™ La unión promiscua de los péptidos virales a HLA-B27 no se correlaciona con su antigenicidad e inmunogenicidad en el contexto de subtipos particulares. El polimorfismo de HLA-B27, probablemente influye en la inmunogenicidad peptídica modulando la estabilidad más que la afinidad. El motivo canónico Arg2 no es necesario para mantener la estructura antigénica de los epítopos peptídicos analizados. ™ Las restricciones al número de ligandos naturales de B*2705 se basan en gran medida en la coexistencia compensada de residuos óptimos y subóptimos en las posiciones de anclaje P1, y sobre todo P3 y P9. Éstas posiciones muestran una contribución jerárquica y aditiva a la afinidad del péptido. Entre los ligandos naturales de longitud no canónica es necesaria la presencia de buenos anclajes en las tres posiciones estudiadas. ™ Los análogos de péptidos antigénicos modificados en su región central mediante espaciadores no peptídicos de tipo bifuncional pueden mejorar la afinidad de unión a la molécula de clase I.

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VIII. ANEXOS

ANEXOS

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VIII. ANEXOS

-Anexo 1-

-Anexo 5-

Galocha B., Lamas, J.R., Villadangos J.A., Albar J.P., López de Castro J.A. (1996). Binding of peptides naturally presented by HLA-B27 to the differentially disease-associated B*2704 and B*2706 subtypes, and to mutants mimicking their polymorphism. Tissue Antigens. 48: 509518.

Lamas, J.R., Paradela, A., Roncal, F., López de Castro, J.A. (1999). The peptide specificity of HLA-B27 subtypes differentially associated to ankylosing spondylitis is modulated at multiple anchor positions. (aceptado en Arthritis and Rheumatism).

-Anexo 2-

-Anexo 6-

García, F., Galocha, B., Villadangos, J.A, Lamas, J.R., Albar, J.P., Marina, A., López de Castro, J.A. (1997). HLA-B27 (B*2701) specificity for peptides lacking Arg2 is determined by polymorphism outside the B pocket. Tissue Antigens. 49: 580-587.

Krebs, S., Lamas, J.R., Poenaru, S., Folkers, G., López de Castro, J.A., Seebach, D., Rognan, D. (1998). Substituting nonpeptidic spacers for the T Cell Receptor-binding part of class I Major Histocompatibility Complex-binding peptides. J.Biol. Chem. 273: 19072-19079.

-Anexo 3-

-Anexo 7-

Rognan, D., Krebs, S., Kuonen, O., Lamas, J.R., López de Castro, J.A., Folkers, G. (1997). Fine specificity of antigen for two clas I major histocompatibility protein alleles (B*2705 and B*2703) differing in one amino acid. J.Comput. Aid. Mol. Des. 11: 463-478.

Poenaru, S., Lamas, J.R., Folkers, G., López de Castro, J.A., Seebach, D., Rognan, D. (1998). Nonapeptide analogues containing (R)-3-hydroxybutanoate and βhomoalanine oligomers: synthesis and binding affinity to a class I MHC protein. (enviado a J. Med. Chem).

-Anexo 4-

-Anexo 8-

Lamas, J.R., Brooks, J.M. Galocha, B. Rickinson, A.B., López de Castro, J.A. (1998). Relationship between peptide binding and T-cell epitope selection: a study with subtypes of HLA-B27. International Immunology. 10: 259-266.

García, F., Rognan, D., Lamas, J.R., Marina, A., López de Castro, J.A. (1998). An HLA-B27 polymorphism (B*2710) that is critical for T-cell recognition has limited effects on peptide specificity. Tissue Antigens. 51:1-9.

ANEXO -1-

ANEXO -2-

ANEXO -3-

463

Journal of Computer-Aided Molecular Design, 11 (1997) 463–478. KLUWER/ESCOM © 1997 Kluwer Academic Publishers. Printed in The Netherlands. J-CAMD 410

Fine specificity of antigen binding to two class I major histocompatibility proteins (B*2705 and B*2703) differing in a single amino acid residue* Didier Rognana,**, Stefan Krebsa, Oliver Kuonena, José R. Lamasb, José A. López de Castrob and Gerd Folkersa a Department of Pharmacy, Swiss Federal Institute of Technology, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland Centro de Biologia Molecular Severo Ochoa, Universidad Autonoma de Madrid, Facultad de Ciencas, E-28049 Madrid, Spain

b

Received 10 March 1997 Accepted 26 May 1997 Keywords: MHC; HLA-B27; Drug design; Molecular dynamics simulations

Summary Starting from the X-ray structure of a class I major histocompatibility complex (MHC)-encoded protein (HLA-B*2705), a naturally presented self-nonapeptide and two synthetic analogues were simulated in the binding groove of two human leukocyte antigen (HLA) alleles (B*2703 and B*2705) differing in a single amino acid residue. After 200 ps molecular dynamics simulations of the solvated HLA–peptide pairs, some molecular properties of the complexes (distances between ligand and protein center of masses, atomic fluctuations, buried versus accessible surface areas, hydrogen-bond frequencies) allow a clear discrimination of potent from weak MHC binders. The binding specificity of the three nonapeptides for the two HLA alleles could be explained by the disruption of one hydrogen-bonding network in the binding pocket of the HLA-B*2705 protein where the single mutation occurs. Rearrangements of interactions in the B pocket, which binds the side chain of peptidic residue 2, and a weakening of interactions involving the C-terminal end of the peptide also took place. In addition, extension of the peptide backbone using a β-Ala analogue did not abolish binding to any of the two HLA-B27 subtypes, but increased the selectivity for B*2703, as expected from the larger peptide binding groove in this subtype. A better understanding of the atomic details involved in peptide selection by closely related HLA alleles is of crucial importance for unraveling the molecular features linking particular HLA alleles to autoimmune diseases, and for the identification of antigenic peptides triggering such pathologies.

Introduction Major histocompatibility complex (MHC)-encoded class I proteins play a major role in the immune surveillance of intracellular pathogens by presenting antigenic peptides to cytotoxic T-lymphocytes (CTLs) at the surface of infected cells [1]. In the last decade, tremendous research efforts have been made to delineate the molecular aspects of antigen presentation to CTLs. Three major breakthroughs in this field were the description of the first class I MHC crystal structure [2], the identification of allele-specific motifs for naturally bound peptides [3], and the prominent role played by MHC-encoded TAP heterodimers for antigen transport [4]. Starting from these three major observations, experimental data on antigen *Dedicated to Prof. D. Seebach on the occasion of his 60th birthday. **To whom correspondence should be addressed.

presentation to MHC proteins are presently accumulating at an incredible pace. The huge amount of molecular details have made human leukocyte antigen (HLA) proteins very interesting drug design targets for two reasons. First, it is likely that the three-dimensional (3D) structures of all class I and class II alleles are very similar, and that the MHC binding mode is rather conserved for most of the presented peptides, within each HLA class [5,6]. Reliable 3D pictures of MHC–peptide complexes are now accessible and can be interpreted with respect to biological data [7– 10]. Second, the expression of certain MHC alleles is associated with either resistance or susceptibility to human immunological diseases like ankylosing spondylitis [11], diabetes [12], rheumatoid arthritis [13] or malaria [14]. One of the strongest linkages known to date between

464 the expression of one class I HLA allele and susceptibility to a pathology is that of HLA-B27 to inflammatory diseases of the joints called spondyloarthropathies [15,16]. For example, more than 95% of patients suffering from ankylosing spondylitis are HLA-B27 positive, while this MHC type is only expressed in 7% of the healthy population [11]. Interestingly, among the 11 HLA-B27 alleles (HLA-B*2701 to HLA-B*2711) reported to date, at least two are not associated with spondyloarthropathies: HLAB*2706 [17,18] and HLA-B*2709 [19]. All subtypes differ among each other only at a few residues, mostly located in the peptide binding groove. Deciphering the molecular parameters responsible for the binding of antigenic peptides to the different HLAB27 subtypes is an absolute prerequisite for better understanding their differential association with susceptibility to spondyloarthropathy, and to identify the sequence of potential arthritogenic peptides [15] that may trigger the disease. Sequencing of peptides naturally presented by the different alleles [20–26] shows that Arg at position 2 (P2) is a conserved motif for peptides binding to all subtypes. Gln2 is an additional motif among B*2701-bound peptides [25], but is a suboptimal residue (not found in vivo) for other subtypes [27–30]. Two other anchoring positions could be disclosed from the frequency of occurrence of amino acids in peptides naturally bound to B*2705. Hydrophobic amino acids are preferred at P3, and hydrophobic as well as positively charged residues can be found at P9. Other positions are more variable and probably indicate a less important binding role. These observations are compatible with the crystal structure of one allele (HLA-B*2705) in complex with a peptide pool [31,32]. As for all other class I alleles crystallized to date, bound ligands are mainly nonapeptides strongly hydrogenbonded in a sequence-independent manner at their Nand C-termini to both ends of the MHC binding groove. The central part of the peptide (from P4 to P8) bulges out of the binding groove (Fig. 1) [32]. Some peptide side chains (P2, P3 and P9) are responsible for the allele specificity of the recognition process, by binding to complementary pockets of the MHC. Notably, the conserved Arg2 of B27-bound peptides is perfectly centered in a polar subsite (pocket B) composed of MHC polymorphic residues (Tyr7, His9, Thr24, Glu45, Cys67). HLA-B*2705 is the only B27 subtype that has been crystallized up to now. Furthermore, although peptide binding motifs have been described for various HLA-B27 subtypes, the exact peptide repertoire selected by each allele is still under investigation, and the exact contribution of individual amino acids at the nine peptide positions to the various subtypes remains largely unknown. HLA-B*2703 contains a single point mutation (Tyr59→His) in a subsite (pocket A) responsible for the binding of the peptide N-terminal residue. HLA-B*2703 probably selects a subset of peptides presented by the

common HLA-B*2705 allele [33,34]. Positively charged residues (Lys, Arg and His) at P1 have been proposed to be one of the main characteristics of this subset common to both subtypes as the replacement of Ser1 by Arg restored the binding of a B*2705-restricted viral epitope to HLA-B*2703 [35]. In addition, basic residues were predominant among self-peptides naturally bound to B*2703 [23]. Additional interactions given by basic side chains could compensate for the weaker interaction of the peptide N-terminus to B*2703 pocket A. However, this is not a stringent requirement because amino acids other than Arg/Lys at P1 (Ala for example) are compatible with a good binding to this allele [28]. To rationalize these data, molecular dynamics (MD) simulations of both alleles in complex with three different peptides were undertaken to delineate similarities and differences in MHC binding, which may explain specificity variations.

Materials and Methods Coordinates setup Starting coordinates were taken from the crystal structure of HLA-B*2705, solved at 2.1 Å resolution [32] and deposited in the Brookhaven Protein Databank [36] with the entry 1HSA. In order to save computational time, only the antigen-binding α1–α2 domains were taken into account in the study. This approximation was previously shown not to alter the accuracy of MD simulations [9,37,38] because only limited interactions exist between the α1-α2 part and the other two domains (α3 and β2m) that do not significantly contact antigenic peptides in the binding groove. This observation has been validated by the crystal structure of one class I MHC protein lacking the membrane-proximal α3 domain, for which a conserved 3D fold of the α1–α2 antigen-binding domain was reported [39]. The C-terminal residue of the α2 domain (Thr182) was protected by an N-methyl group to avoid unrealistic electrostatic interactions. The HLA-B*2703 subtype was obtained from the HLA-B*2705 crystal structure by mutating Tyr59 into His, without changing the direction of the side chain. All MHC-bound nonamers were built from the peptide (ARAAAAAAA) modeled in the original crystal structure [32] by substituting the corresponding residue for alanine without altering the direction of the side chains. Six crystal water molecules were explicitly taken into account, as they are located in the peptide binding cleft and bridge the binding of the peptide to the protein X-ray structure. Polar hydrogen atoms were then added and the complexes were centered in a 7.5 Å thick shell of TIP3P water molecules [40] without positional constraint on solvent atoms. Any water atom closer than 1.75 Å to any solute atom was discarded, so that approximately 1500 water molecules were added to each MHC–peptide binary complex.

465 Parametrization of the N-terminal b-alanine monomer The N-terminal β-alanine was parametrized for AMBER 4.0 [41] using a previously described procedure [42]. Briefly, atomic coordinates for β-alanine were obtained from the SYBYL biopolymer dictionary [43] and optimized by semiempirical quantum mechanics (MOPAC 6.0) using the PM3 Hamiltonian [44]. Potential-derived atomic charges were then computed on this geometry by a single-point SCF calculation at the MNDO level [45]. As new atom types were not defined, nonexisting force constants have been assigned according to available AMBER values for closely related bond, angle and dihedral types. They were incorporated into the parm91 parameter set. Molecular mechanics and dynamics simulations All computations were performed on a CRAY J90 using the AMBER 4.0 program [41] and the united-atom representation of the parm91 parameter set. Since explicit water molecules were taken into account, a dielectric constant of 1 was used for all calculations. To avoid splitting dipoles, nonbonded interactions were calculated within a residue-based cutoff of 10 Å. The solvent atoms were first relaxed by 1000 steps of steepest descent energy minimization, the solute being held fixed. The solvated complex was then fully minimized by 1000 steps of steepest descent, followed by a conjugate gradient minimization procedure until the rms gradient of the potential energy was less than 0.25 kcal mol−1 Å−1. The minimized coordinates were thereafter used as a starting point for an MD simulation at constant temperature. Initial velocities were taken from a Maxwellian distribution at 50 K and an integration step of 2 fs was used. The system was progressively heated from 50 to 297 K during the first picosecond, the temperature being held at 297 K for the rest of the simulation by coupling the system to a heat bath [46] using a temperature coupling constant of 0.05 ps. All bond lengths were constrained to their equilibrium values using the SHAKE algorithm [47] with a bond length tolerance of 2.5 × 10−4 Å. Coordinates, energies and velocities were collected and saved every 250 steps (0.5 ps) for 200 ps. The analyses of MD trajectories were achieved using in-house routines and the CARNAL module of AMBER [41]. Calculation of electrostatic interaction energies Electrostatic free energies were computed by solving the linear form of the Poisson–Boltzmann equation using the finite-difference method [48,49] of the DelPhi program [50,51]. Peptides, MHC proteins and MHC–peptide complexes were centered in three-dimensional boxes with resolutions of 2.0, 1.20 and 1.10 grid points per Å, respectively. For each calculation, 90% of the box was filled with the corresponding molecule. Atomic radii and charges were taken from the AMBER 4.0 united-atom

parameter set [41]. Inner and outer dielectrics were assigned values of 2.0 and 1.0 (vacuum) or 80 (water environment). An ionic strength of 0.145 M and an ion exclusion radius (Stern layer) of 2.0 Å were used according to previously reported solvent calculations [51]. A probe radius of 1.8 Å was utilized for computing the surface at which the electrostatic potential was extrapolated. Peptide synthesis Peptides 1 and 3 were synthesized as previously described [28]. Peptides 2 and 4 were synthesized by automated, multiple solid-phase peptide synthesis with a robot system (Syro, MultiSynTech, Bochum, Germany) using an Fmoc/tBu strategy. For side-chain protection, Tyr(tertbutyl), Ser(tert-butyl), Thr(tert-butyl), Glu(tert-butyl), Gln(trityl), Arg(2,2,3,5,5-pentamethyl-chromansulfonyl) and Lys(tert-butyloxy-carbonyl) were used. The N-terminal residues were obtained using single couplings with diisopropylcarbodiimide/1-hydroxy-benzotriazole activation, 10-fold excess and a coupling time of 1 h on the 2chlorotritylchloride resin. The peptides were cleaved with trifluoroacetic acid/thioanisole/thiocresol (20:1:1) within 3 h, collected by centrifugation and lyophilized from water. They were then purified by reversed-phase HPLC (Merck-Hitachi, Darmstadt, Germany) on a nucleosil 5 µM/C18 column (125 × 3 mm) at a flow rate of 600 µl/min. The absorbance was measured at 220 nm. The solvent system used consisted of 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B). A linear gradient from 10 to 60% B in 30 min was applied. The peptides were purified to homogeneity by a second HPLC on a versapack 10 µm C18 column (300 × 7.8 mm) at a flow rate of 2 ml/min with the same buffer system, and a linear gradient from 0 to 40% B in 35 min followed by a 40–60% B linear gradient for 20 min. Furthermore, the peptides were analyzed by ion spray mass spectrometry on a triple-quadrupole mass spectrometer API III with a mass range of m/z = 10–2400 equipped with an ion spray interface (Sciex, Thornhill, ON, Canada), and quantified by amino acid analysis using a 6300 amino acid analyser (Beckman, Palo Alto, CA, U.S.A.). Peptide binding assay The quantitative assay used has been described previously [29]. Briefly, RMA-S transfectants expressing B*2705 or B*2703 were used. These are murine cells with impaired TAP-mediated peptide transport and low surface expression of (empty) class I MHC molecules, which can be induced at 26 °C [52] and stabilized at the cell surface through the binding of exogenously added peptides. These cells were incubated at 26 °C for 24 h. After this, they were incubated for 1 h at 26 °C with 10−4–10−9 M peptides, transferred to 37 °C and collected for flow

466

B C P1

P6

P4

P2

P8

P9

P5 P7

A P3

F E D

Fig. 1. Orientation of a canonical nonapeptide (ball-and-stick model) in the binding groove of HLA-B27 (cyan ribbons) [32]. Peptide positions are labeled at the Cα atoms from 1 (P1) to 9 (P9). MHC specificity pockets (A to F) are displayed according to the usual nomenclature [72]. The figure has been obtained with the MOLSCRIPT [73] and Raster3D [74] programs.

microcytometry (FMC) analysis with the ME1 mAb (IgG1, specific for HLA-B27, B7 and B22) [53] after 4 h for B*2705, or after 2 h for B*2703. The determinant recognized by ME1 is not affected by bound peptides or by HLA-B27 polymorphism (data not shown). The binding of a given peptide was measured as its C50. This is its molar concentration at 50% of the fluorescence obtained with that peptide at 10−4 M. Peptides with C50 ≤ 5 µM were considered to bind with high affinity, as these were the values obtained for most of the natural B27-bound ligands. C50 values between 5 and 50 µM were considered to reflect intermediate affinity. C50 ≥ 50 µM indicated low affinity. Peptides having C50>100 µM were assumed not to bind. The binding of peptide analogues was measured as the concentration of the peptide analogue required to obtain the fluorescence value at the C50 of the unchanged peptide. This was designated as EC50. Relative binding was expressed as the ratio between the EC50 of the peptide analogue and the C50 of the corresponding unchanged peptide.

histone peptide was also studied, in order to investigate the influence of pocket B–P2 interactions that may also participate in the peptide discrimination [28]. Dynamical properties of MHC–peptide complexes Monitoring instantaneous rms deviations (rmsd’s) of protein atoms from the starting structure is usually performed to ascertain the reliability of MD simulations [54]. For both alleles complexed to peptides 1–3, similar rmsd values were observed (about 1.7 Å for backbone atoms; TABLE 1 RELATIVE BINDING OF THREE NONAPEPTIDES TO TWO HLA-B27 SUBTYPES Peptide

1 2 3 a

Results and Discussion A naturally bound nonapeptide (1) from the human histone H3 protein was taken as reference for its equal binding to both subtypes (Table 1). The Ala1 analogue (2) was chosen for its unexpected high affinity for both subtypes [28,35]. Finally, the Gln2 analogue 3 of the human

b

Sequence

RRYQKSTELb ARYQKSTEL RQYQKSTEL

Relative bindinga B*2703

B*2705

1 (2×10−6) 1.5 >>100

1 (2×10−6) 2 10

Data are expressed as the molar excess of peptide analogue, relative to the wild-type peptide 1, at which HLA-B27 fluorescence (measured by FMC analysis with an anti-B27 monoclonal antibody) on RMA-S cells was half the maximum obtained with peptide 1. The molar concentration of peptide 1 at 50% maximum fluorescence (C50) is given in parentheses. Human histone H3 peptide: a self-peptide, naturally bound to HLAB*2705 [20] and to B*2703 [23]. Dominant anchor residues are displayed in boldface.

467 data not shown), indicating that protein distortion upon ligand binding cannot account for the different binding affinities of these peptides. Stable rmsd values (1–1.5 Å) were also observed for bound peptides. Interestingly, they remain in the same range as that observed for different peptides co-crystallized with the same MHC host protein [55,56]. One exception concerns the weakest binder (peptide 3 to HLA-B*2703), for which larger distortions (up to 2.5 Å) were observed and still increasing after 200 ps simulation. To follow a possible dissociation of peptides 1–3 from the two HLA-B27 alleles, the distance between ligand and protein center of mass (cmass) was monitored. For binding peptides (1, 2), whatever the B27 subtype, this intermolecular distance increases during the warm-up phase from 8 to 8.5 Å and remains constant for the rest of the simulation (Fig. 2). The weak binder (peptide 3) exhibits a slight but continuous increase of the intermolecular distance, suggesting a partial dissociation of the ligand from the binding groove. This phenomenon was significantly enhanced for the less stable complex (3a: peptide 3 in complex with HLA-B*2703). However, localization of the dissociating peptide amino acids is not possible with this analysis. For example, TcR-binding residues may bulge even more out of the peptide binding site and induce similar shifts in the intermolecular distance between center of masses. Therefore, this analysis was extended by computing the cmass of bound peptide substructures (MHC anchors: P1, P2, P3, P9; TcR anchors: P4, P5, P6, P7, P8) for each MD conformation and the distance relating it to the 1a, 2a, 3a,

10.0

protein cmass (Table 2). An examination of d2 and d3 intermolecular distances for the weak binder 3 clearly shows that MHC-anchoring amino acids only are progressively expelled from the binding groove. The dissociation is more significant when compound 3 is complexed to HLA-B*2703, which relates well with the observed binding data (Table 1). The TcR-binding region (P4–P8) is similarly bulging out (at least quantitatively) of the binding cleft for all the six studied complexes (d3 distance, Table 2). An even more precise analysis has been done by evaluating the inter-cmass distance between individual MHC anchor residues and their complementary pocket (A, B, D and F; d4–d7 distances, Table 2). Surprisingly, the repulsion noticed for peptide 3 in complex with both alleles seems to be located at the P9–pocket F interaction level (d7 = 7.3 Å), far from where protein and peptide single-point mutations occur. Interestingly, the critical d5 distance, illustrating the strength of the most important interaction between the invariant Arg2 and pocket B, is higher (5.1 ± 0.4 Å) for the less stable complex (3a) than for other pairs (Table 2). The different d4 distances reported here, especially for peptides 1 and 3 and peptide 2, are related to the size of the corresponding P1 side chain (Arg versus Ala). It indicates that the Arg1 side chain is pointing away from its binding subsite (pocket A) and therefore induces higher d4 inter-cmass distances. However, for a given P1 side chain, higher d4 distances are always observed for HLA-B*2703. This indicates that the Tyr59→His mutation found in the latter allele is detrimental for a stable and strong binding of P1 side chains to pocket A of the protein. The highest stan-

1b 2b 3b

Distance, Å

9.5 9.0 8.5 8.0 7.5 7.0 0

50

100

150

200

Time, ps Fig. 2. Time course of the distance (in Å) between the center of mass of peptides 1–3 and that of the host HLA-B27 subtype (a: B*2703; b: B*2705).

468

2.0 1a, 2a, 3a,

1.8

1b 2b 3b

1.6

rmsf, Å

1.4 1.2 1.0 0.8 0.6 P1

P2

P3

P4

P5

P6

P7

P8

P9

Pn Fig. 3. Rms atomic fluctuations of peptides 1–3 when bound to an HLA-B27 protein (a: B*2703; b: B*2705). Pn represents the peptide position (from 1 to 9).

dard deviations are found for the P3–pocket D interaction (d6, Table 2). The 0.6 Å variations, observed in such nonbonded distances, correspond to the alternative establishment of strong and weak hydrophobic contacts, which may be explained by the topology of the binding groove. Pocket D is a hydrophobic subsite open to the central part of the binding cleft, and partially filled by hydrophobic side chains [20]. The significantly higher variance of the d6 distance (Table 2) suggests that this interaction is the most flexible one and that different local conformations at P3 are compatible with a good occupancy of pocket D. Retrospectively, it explains why the P3–pocket D interaction may be important for an optimal peptide binding to HLA-B*2705. Bulky hydrophobic nonnatural side chains (α- and β-naphthylalanine, cyclohexylalanine) have been shown to significantly enhance binding to the

HLA-B*2705 protein [38,57]. This positive effect probably results from two correlated components: (i) the existence of additional contacts to pocket D, a pure enthalpic effect; and (ii) a reduced flexibility of the MHC-bound P3 side chain, a favorable entropic effect. Atomic fluctuations Atomic fluctuations of MHC-bound peptides were computed from mean conformations, time-averaged over the last 50 ps (Fig. 3). As expected, MHC anchors are much less flexible than the TcR-binding middle part. If atomic mobility of the bound ligand is considered, it is not possible to depict real differences in the binding of peptides 1 and 2 to the two subtypes. However, the C-terminal anchor residue clearly tends to be more flexible when the corresponding ligand does not strongly bind to the B27

TABLE 2 DISTANCE BETWEEN PROTEIN AND PEPTIDE CENTER OF MASSES Distance (Å)

Peptide 1a

d1 d2 d3 d4 d5 d6 d7

08.2 05.9 11.5 03.6 04.7 04.9 02.5

1b ± ± ± ± ± ± ±

0.3 0.3 0.4 0.3 0.2 0.6 0.4

08.5 05.9 12.1 02.9 04.7 04.8 02.6

2a ± ± ± ± ± ± ±

0.3 0.3 0.5 0.4 0.3 0.5 0.3

08.3 05.5 12.3 02.3 04.7 05.3 02.9

2b ± ± ± ± ± ± ±

0.3 0.3 0.5 0.3 0.3 0.5 0.3

08.2 05.6 10.7 01.5 04.4 05.0 03.4

3a ± ± ± ± ± ± ±

0.4 0.4 0.5 0.5 0.3 0.7 0.4

09.3 07.3 12.0 03.4 05.1 04.6 07.3

3b ± ± ± ± ± ± ±

0.9 0.5 0.6 0.4 0.4 0.5 0.6

09.1 06.6 12.3 02.8 04.7 05.2 04.8

± ± ± ± ± ± ±

0.7 0.5 0.6 0.3 0.3 0.6 0.5

d1: protein–peptide; d2: protein–MHC anchors (P1–P3, P9); d3: protein–TcR anchors (P4–P8); d4: pocket A–P1; d5: pocket B–P2; d6: pocket D–P3; d7: pocket F–P9.

469

2.0 1a, 2a, 3a,

1b 2b 3b

Accessible / Buried

1.5

1.0

0.5

0.0 P1

P2

P3

P4

P5

P6

P7

P8

P9

Pn Fig. 4. Ratio of accessible to buried surface area for relaxed MD time-averaged conformations. Surface areas were calculated with the MS program [75] using a 1.4 Å probe radius. High ratios were truncated to a value of 2.0. Pn represents the peptide position (from 1 to 9).

subtype. The highest flexibilities are observed for the weakest binder (peptide 3 to B*2703), especially at the important anchoring positions (P2, P3 and P9). It is logical to find that the mutation at P2 (for peptide 3)

weakens the interactions to MHC pocket B that has been designed to accommodate an arginine side chain [32]. However, the P2 amino acid is more flexible when the corresponding peptide is complexed to HLA-B*2703

30 >50% 25-50%

H-bonds Number

25

20

15

10

5

0 1a

1b

2a

2b

3a

3b

Complex Fig. 5. MHC–peptide H-bonding frequency for peptides 1–3 in complex with HLA-B27 subtype (a:B*2703; b: B*2705). H-bonds have been geometrically defined by an acceptor (A) to donor (D) distance less than 3.25 Å and a D-H..A angle greater than 120°. Interactions were statistically monitored throughout the simulations for a total of 400 conformations per MHC–peptide complex. Two categories of H-bonds were defined: strong ones with frequencies higher than 50% and medium ones with occurrences between 25 and 50%.

470

Glu45

Thr24

Tyr59 His59

His9

Glu63

P1

P2 Tyr7

Tyr171

Tyr99 Trp167

Met5

Tyr159

Fig. 6. Crystal structure of HLA-B*2705 [32]. The view is focused on MHC pocket A (Met5, Tyr7, Tyr59, Glu63, Tyr159, Trp167, Tyr171) and pocket B (His9, Thr24, Glu45, Tyr99) side chains interacting via H-bonds (direct H-bonds: green broken lines; water-mediated H-bonds: yellow broken lines) with the P1–P2 positions of a bound peptide. The following color coding has been used: carbon, white (protein) or orange (peptide); nitrogen, blue; oxygen, red; sulfur, yellow. Bound water molecules are shown as cyan balls. Arrows indicate the direction of the H-bonds (from the donor to the acceptor). HLA-B*2703 was obtained by mutating Tyr59 into His (SYBYL Biopolymer module) [43]. The side-chain χ2 dihedral was just modified here in order to bring the Nε atom as close as possible to the bound water molecule. However, whatever the rotamer chosen, a direct H-bond to a water molecule is not possible. The interatomic distance between the peptide N-terminus and Tyr59 (OH) or His59 (Nε2) atoms is 4.31 and 5.35 Å, respectively. Figures 6–8 have been obtained by using the rendering program Raster 3D [74].

(compare 3a and 3b, Fig. 3). Again, the most striking differences are not observed at the variable amino acids of the peptides (P1, P2) but at the C-terminal anchor (P9), a feature already noticed for other MHC–peptide complexes [9]. One may hypothesize that the N-terminal tripeptide (P1-P2-P3) determines the stability of peptide– MHC interactions over the whole length of the binding groove by controlling the conformational space accessible to the bulging middle part and, consequently, the binding capacity of the following C-terminus. However, atomic flexibilities of the P1–P3 and P4–P8 parts are not interrelated. It is also possible that these differences are related to the short time scales (200 ps) used for simulating the complexes and that much longer simulations are needed to see significant molecular differences at positions P1 and P2 of the peptide ligand. Nanosecond MD simulations of macromolecules are nowadays feasible [58,59], but still remain unrealistic as a structure–activity relationship tool for comparing a series of ligands in their protein-bound state. Accessible versus buried surface areas Whether peptide flexibility correlates with dissociation from the binding cleft was addressed by looking at access-

ible and buried surface areas of each HLA-bound peptide residue (Fig. 4). Only position 9 of peptide 3 in complex with B*2703 was much more accessible than the others. Otherwise, the main anchor P2 was similarly buried whatever the peptide and the host HLA protein. This means that a partial dissociation was only observed for one position (P9) in one complex (3a) and that the previously reported higher flexibility of Gln2 for the same peptide– MHC complex did not correspond with a release of the P2 side chain from pocket B. It may however influence the quality and frequency of hydrogen bonds between the Gln2 side chain and pocket B of both B27 alleles. The influence of secondary anchor positions is more difficult to ascertain. P3 is similarly buried for all MHC–peptide complexes. P6 (Ser) and P7 (Thr) positions are probably accessory anchor positions that marginally bind, in some conformations, to the central part of the peptide binding groove (pockets C/E). This observation is not incompatible with the high atomic fluctuations of P6-P7 amino acids (Fig. 3), as their side chains are directed towards the binding groove but without reaching its floor. P4 (Gln), P5 (Lys) and P8 (Glu) residues are potential candidates for TcR recognition because of their concomitant atomic flexibility and surface accessibility.

471

Glu63

His59

Glu45

Thr24 P2

Tyr7

P1

Tyr159

Trp167 Tyr171

His9

Tyr99

Met5

Fig. 7. MD model of complex 3a, focused on MHC pockets A–B (see the legend to Fig. 6). A mean conformation was averaged from the last 100 conformers and submitted to 500 steps of steepest descent, followed by 1500 steps of conjugate-gradient energy relaxation.

Qualitative and quantitative analysis of peptide–MHC Hbonds Reporting the number of MHC–peptide H-bonds as well as their frequencies during the simulation allows a

Tyr59

clear distinction between peptides 1 and 2 and the Gln2 analogue (peptide 3, Fig. 5). The stability of protein– peptide H-bonds was assessed by computing the frequency of occurrence of the interaction throughout the MD tra-

Glu45 Glu63 Thr24 His9 P2 P1

Tyr7

Glu163 Tyr171

Trp167

Met5

Tyr99 Tyr159

Fig. 8. MD model of complex 3b, focused on MHC pockets A–B (see the legend to Fig. 6). The mean conformation was obtained as described in Fig. 7.

472 TABLE 3 MHC–PEPTIDE H-BONDS WITH A FREQUENCY HIGHER THAN 50% Pn atom

HLA-B27

P1

Tyr7 (OH)

N

59

His (NE2)

1a

1b

2a

1 1

2b

1

1

1 1

1

(OE2)

NE

Glu

Glu63 (OE1)

1

Glu63 (OE2) O P2

N

1

Tyr159 (OH) 63

Glu (OE1) Glu45 (OE2)

×

×

×

×

3

×

×

3

1

1

1

3 3

×

×

×

×

OE1

Tyr (OH)

×

×

×

×

3

NE2

His9 (NE2)

×

×

×

×

3

Glu45 (OE2)

×

×

×

×

NH1

99

His9 (NE2)

1

Thr (OG1) NH2

Thr (OG1)

1

Glu45 (OE1)

1

Glu45 (OE2)

1

24

P3

N

Tyr (OH)

P6

OG

Ala69 (O)

1 1 1

1

1

OE1

Lys

N

Asp77 (OD1)

OXT

1

1

Tyr (OH)

1 1

(NZ)

×

1

×

×

×

×

1

×

×

×

×

1

1

1

1

(OE1)

Thr143 (OG1) Lys

×

1 1

84

146

×

1

Trp147 (NE1) 146

3 ×

1

1

Thr73 (OG1) O

1

3

Glu63 (OE1) 99

1 3

24

P9

1

1 1

Glu63 (OE1)

P8

1

1

Glu63 (OE2) NE

1 3

Glu163 (OE2)

NH2

1

1

Glu63 (OE2) 163

3b

3

Glu45 (OE2) Glu63 (OE1)

3a

1

1

1

1

1

1

1

1

1

Empty boxes indicate interactions that are common to at least two complexes, whereas filled boxes represent unique MHC–peptide hydrogen bonds. The absence of a specific side chain is featured by a cross.

jectory (400 conformations). A frequency higher than 50% was chosen to characterize strong H-bonds. Medium interactions were assigned a frequency between 25 and 50%. About 25 H-bonds have been identified for peptides 1 and 2 in complex with B*2703 and B*2705 while 50% less could be found for the Gln2 analogue with the two subtypes (Fig. 5). The distribution of strong and medium Hbonds correlates well with the binding potency of the peptide. A similar number of strong H-bonds were found

for complexes 1a, 1b, 2a and 2b, consistent with the similar binding efficiencies of peptides 1 and 2 to both subtypes. On the other hand, a reduced number of medium and/or strong H-bonds (peptide 3 in complex with the two alleles) correlates with the decreased binding of this peptide. The weakest binding potency (peptide 3 to B*2703) could effectively be qualitatively and quantitatively related to the distribution of intermolecular Hbonds. Not only the number but also the quality of the MHC–ligand interactions correlates well with the binding potency. To accurately localize the interactions that may explain peptide specificity variations, all H-bonds with frequencies higher than 50% were identified for the six complexes (Table 3). The first noticeable difference between the two HLA-B27 alleles is the H-bonding network between the MHC residues involved in binding to the peptide P1 position. In HLA-B*2705, two amino acid side chains are H-bonded to the peptide N-terminus (Tyr7/Tyr171 in the crystal structure, Tyr7/Glu63 in the MD models) (Table 4, Figs. 6–8). Both side chains are fixed by a subtle waterrelayed H-bond network involving proximal MHC side chains (Tyr59, Glu45, Tyr171). Tyr59 is directly bound to Tyr171, and indirectly to Tyr7, Glu45 and Glu63. The single point mutation occurring for HLA-B*2703 (Tyr59→His) perturbs this network. The bound water molecule disappears and the peptide N-terminus binds to His59 and no more to Tyr7 (Table 3, Fig. 7). The consequence on the H-bond balance is a loss of one direct MHC–MHC interaction (His59 cannot interact with Tyr171) and five watermediated interactions for complex 3a (Fig. 7). The resulting conformational change may be well accommodated as far as P2 is strongly bound to pocket B (His9, Thr24, Glu45) and the resulting H-bonds are strong enough to maintain the peptide in the binding groove (P2=Arg). If P2 is not an arginine (peptide 3), the resulting interaction to pocket B (Thr24 and Glu45 notably) is much weaker and the conformational rearrangement at P1 is important (see the three new H-bonds for the P1 position in complex 3a, Fig. 7). The Arg to Gln change at the P2 position of the bound peptide is better tolerated by HLAB*2705 (complex 3b, Fig. 8) as the Tyr59 side chain is still able to fix the position of Tyr7. During the MD simulation, the N-terminal Cα-N bond of the bound peptide has rotated to gain a new H-bond to Glu63. However, it is still bound to Tyr7 as in the reference structure. Importantly, the Gln2 side chain is bound to Glu45 and Tyr99, thus providing additional interactions to pocket B when compared to complex 3a (Fig. 8). The quality of the interaction between Gln2 and pocket B is, however, much inferior to that observed for peptide analogues 1 and 2 bearing an optimal Arg residue (Table 4), thus explaining the reduced binding affinity of peptide 3 for HLAB*2705. For the set of peptides studied here, the advantage of

473

2.0

2a 2b 4a 4b

Accessible/buried

1.5

1.0

0.5

0.0 P1

P2

P3

P4

P5

P6

P7

P8

P9

Pn Fig. 9. Accessible versus buried surface areas of peptides 2 and 4 in complex with B*2703 (a) and B*2705 (b) alleles (see the legend to Fig. 4).

Arg over Ala at P1 could be quantified by the gain of two water accessible salt bridges to Glu63/Glu163 (Table 3). However, this does not correspond to a higher binding affinity of the Arg analogue when compared to the Ala1 peptide. An Ala side chain is much easier to desolvate and optimally interacts with conserved apolar residues of pocket A (Met5, Trp167), thus explaining a rather similar binding affinity of peptides 1 and 2 to both subtypes.

However, the present model cannot fully explain recent data, indicating that basic residues are overrepresented at the P1 position of B*2703-bound natural ligands [23]. From a purely statistical point of view, various rotamers of basic P1 side chains could develop a salt bridge with at least three negatively charged amino acids located at the rim of pocket A (Glu58, Glu63, Glu163), and thus stabilize the MHC–peptide complex.

30 > 50% 25- 50%

25

H-Bonds

20

15

10

5

0 2a

2b

4a

4b

Complex Fig. 10. Intermolecular hydrogen bonds for peptides 2 and 4 in complex with B*2703 (a) and B*2705 (b) alleles (see the legend to Fig. 5).

474 TABLE 4 MHC–PEPTIDE INTERACTION ENERGIES CALCULATED FROM THE LINEAR POISSON–BOLTZMANN EQUATION AND AMBER FORCE-FIELD CALCULATIONS Peptide

∆G0coul

1a 1b 2a 2b 3a 3b

−499 −542 −359 −389 −306 −371

a

b

c d e

f g

a

∆G0reac 487 498 340 335 334 380

b

∆G0elec

c

−12 −44 −19 −54 −28 −09

∆Helec

d

−87 −79 −72 −75 −44 −62

∆Hvdw

e

−90 −97 −77 −80 −68 −67

∆Gtot

∆Htot

f

−102 −141 0− −96 −134 0− −40 0− −58

g

−177 −176 −149 −155 −112 −129

0 ∆G0coul: Coulombic component of MHC–peptide electrostatic interaction energy (charge–charge, charge–dipole, dipole–dipole interactions). ∆Gcoul = ∆G0coul(P–L) − ∆G0coul(P) − ∆G0coul(L) [50], where P–L describes the protein–ligand complex, P the protein and L the ligand. ∆G0reac: corrected self-reaction field component of MHC–peptide electrostatic interaction energies (energy required to transfer a molecule from a continuum dielectric (vacuum) to another (water). ∆G0reac = ∆G0reac(P–L) − ∆G0reac(P) − ∆G0reac(L). As the contribution of the protein–ligand complexes (∆G0reac(P–L)) and of the isolated protein (∆G0reac(P)) could be omitted from the calculation without affecting the reliability of the results [61], this component corresponds here to the free energy of peptide desolvation (−∆G0reac(L)). ∆G0elec: total electrostatic interaction energy (∆G0coul + ∆G0reac). ∆Helec: AMBER electrostatic interaction energy (ε = 4rij). ∆Hvdw: AMBER van der Waals interaction energy. ∆Hvdw = A/r12 − B/r6, where r is the distance between atom pairs and A and B are atom-typedependent parameters. ∆Gtot: total interaction energy (∆G0elec + ∆Hvdw). DHtot: total AMBER interaction enthalpy (∆Helec + ∆Hvdw).

MHC–peptide interaction energies Interaction energies were extrapolated for all six energy-minimized time-averaged conformations (Table 4) by summing up the van der Waals nonbonded interaction energy (calculated with the AMBER 4.0 force field) and the electrostatic component (calculated by solving the linear form of the Poisson–Boltzmann equation), as recently described [60]. As both ligands and protein structures are very similar for all complexes, distortion energies as well as translational/rotational entropy losses upon binding were neglected here. Moreover, the self-reaction field energy component of the electrostatic interaction energy was limited to the contribution of the isolated peptide (free energy of desolvation) and calculated from the bound-peptide coordinates extracted from the MHC– peptide binary complexes. It has recently been shown that neglecting the protein contribution to the self-reaction field energy is indeed possible and does not alter the reliability of the obtained results [61]. Our computational protocol is able to properly rank the binding of the three peptides 1–3 to both MHC alleles. Peptide 3 clearly interacts much weaker than the other two peptides 1 and 2, whatever the MHC allele. The weakest interaction energy was observed for binding of 3 to B*2703, and is thus in agreement with binding data (Table 1). Force-field interaction enthalpies (calculated by summing up both AMBER van der Waals and electrostatic components, using a dielectric permittivity of 4rij) were much less related to the observed binding data, as peptide 2 was always disfavoured with regard to peptide 1 (Table 4). Notably, taking into account the peptide desolvation energy by the continuum eletrostatics method permits to compensate for the weakest Coulombic interactions provided by peptide 2 (P1=Ala) to both alleles, with

respect to that observed for the Arg1 analogue. It may be noticed that the free electrostatic interaction energies (∆G0elec, Table 4) computed by the continuum electrostatics method were also in rather good qualitative agreement with the binding data reported in Table 1. Hence, the three peptides are highly polar and interact mainly via Hbonds and salt bridges. For an even more realistic ranking of highly polar ligands than those presented here, free energy perturbation [62] is probably the method of choice. Unfortunately, the enormous amount of CPU time that would be necessary for this computation precludes its systematic use in fast screening of a set of congeneric molecules. Synthesis and in vitro binding assays in this case provided a faster and experimentally determined answer. MD simulation of MHC–peptide complexes could relate observed binding potencies and allele specificity to simple molecular criteria (inter-cmass distances, atomic fluctuations, accessible surface areas, distribution and TABLE 5 INFLUENCE OF A P1 β-AMINO ACID ON THE HLA-B27 SUBTYPE SELECTIVITY OF A MODIFIED HLA-B27 LIGAND Peptide number

2 4 a

b

Sequence

(P1-RYQKSTEL) P1=Ala P1=Balb

EC50 (µM)a B*2703

B*2705

3.0 7.5

04.0 20

Concentration of the peptide (in µM) at which HLA-B27 fluorescence (measured by FMC analysis with an anti-B27 monoclonal antibody) on RMA-S cells was half the maximum obtained with the wild-type peptide (peptide 1, Table 1). Bal: β-alanine (H2N-CH2-CH2-CO).

475

Glu45

His59

Glu63

Thr80

Asp77

Leu95

Thr24

Leu81

Tyr84

His9 Lys5

Gln4

Leu9

Arg2 Ser6 Glu8 Bal1

Thr7

Tyr3

Tyr7

Asp116 Tyr123

Tyr171

Trp167

Thr143

Glu163

Lys146

Tyr99 His114

Tyr159

Trp147

Leu160

Val152

Leu156

Fig. 11. Energy-minimized time-averaged MD model of complex 4a. The MHC protein backbone is displayed as a solid cyan tube, with peptide-interacting side chains. The bound peptide 4 is represented by sticks. The following color coding has been used: carbon, white (protein) or green (peptide); nitrogen, blue; oxygen, red; sulfur, yellow. Bound water molecules are shown as cyan balls. Yellow broken lines indicate MHC–peptide H-bonds.

location of intermolecular H-bonds). A single point mutation in the HLA binding groove is sufficient to break an H-bond network in the vicinity of the peptide N-terminus. As previously suggested on the basis of peptide binding

analyses [28], this minor change strengthens even more the binding role of the dominant anchor P2 side chain (Arg) for one allele (HLA-B*2703) and explains why changing P2 to Gln has more detrimental effects in pep-

Glu45

Tyr59 Glu63

Thr80

Asp77

Thr24

Leu81 Tyr84

Gln4

Leu95

Lys5

Glu8

Ser6

Bal1

Tyr3

Asp116

Tyr7 Ty171

Leu9

Thr7

His9

Arg2

Ty123

Trp167 Tyr99 Glu163

His114 Tyr159

Trp147

Lys146

Thr143

Val152

Leu156

Leu160

Fig. 12. Energy-minimized time-averaged MD model of complex 4b (see the legend to Fig. 11).

476 tide binding for HLA-B*2703 than for HLA-B*2705, where compensatory stabilization of the MHC–peptide complex is still possible by H-bonded MHC side chains. Interestingly, the most spectacular consequence of protein/peptide variability affects an area far away (10 Å) from the location of the point mutations. It concerns the stability of the interaction between the peptidic C-terminal residue and its complementary pocket F, which has recently been shown to play a decisive role in linking particular B27 alleles to spondyloarthropathies [24,26]. Protein-based design Relating the structure of B27 subtypes to the sequence of their naturally bound peptides is a crucial step in identifying potential immunodominant epitopes that may discriminate alleles and confer susceptibility or resistance to autoimmune diseases. One striking feature concerns the single point mutation (Tyr59His) distinguishing HLAB*2705 from HLA-B*2703, which is unique among HLA proteins. It is believed that B*2703 selects a subset of the peptides presented by HLA-B*2705 [34]. Recent studies have identified some peptides that are naturally presented by both subtypes, and at least one natural B*2705 ligand (the undecamer RRYLENGKETL) is not presented by B*2703 [23,63]. The only difference between both alleles concerns the position 59 located in pocket A which interacts with the N-terminal amino acid of the bound peptide (Fig. 6). As pocket A is slightly wider for HLA-B*2703, extending the peptide backbone towards His59 by replacing the natural P1 residue by a β-amino acid should theoretically allow a better discrimination of both alleles. This structural change should be much better accommodated by HLA-B*2703 (H-bond between His59 and the N-terminus of the P1 β-amino acid) than by HLA-B*2705, for which a steric clash with the Tyr59 side chain may be expected. Starting from the self-peptide 1 (RRYQKSTEL) naturally presented by HLA-B*2705 [20] and B*2703 [23], Ala and Bal (β-alanine) were substituted for the natural Arg at P1 (Table 5). The Ala1 peptide analogue was here taken as a reference for its strong binding to both subtypes. The two ligands were synthesized and tested for their binding to B*2703 and B*2705. As expected from the topology of the binding cleft, only the Bal analogue could discriminate between the two subtypes, with a better binding to HLA-B*2703 (Table 5). To rationalize the experimental binding data, the nonnatural ligand 4 was simulated a posteriori, in complex with both alleles, using exactly the same conditions as those employed for simulating the natural MHC–peptide complexes (see the section Computational procedures). Using two of the previously described molecular parameters (accessible versus buried surface area of the bound ligand, intermolecular H-bonds) as quality control of the complex stability, peptide 4 was indeed found to be much better accommodated by B*2703 than by B*2705 (Figs.

9 and 10). Interestingly, the weak binding of the β-Ala peptide to B*2705 could also be related to a partial dissociation of the C-terminus from its complementary pocket F (Fig. 9), far away from the peptide mutation site. The present data, in agreement with previous MD simulations of different MHC–peptide complexes [9], suggest that the expulsion of the C-terminus from pocket F could be the very first event in the dissociation of weak binding peptides from class I MHC binding grooves. The modified P1 position is, however, significantly more buried when the host protein is the B*2703 allele (Fig. 9). A qualitative and quantitative analysis of intermolecular hydrogen bonds also supports the reported binding data. A total of 15 H-bonds could be depicted for complex 4b (peptide 4 in complex with B*2705), whereas 26 interactions have been found for complex 4a (peptide 4 in complex with B*2703, Fig. 10). However, this analysis was unable to explain the reduced affinity of the β-Ala compound for B*2703, when compared to that of the natural Ala analogue 2 (Table 4). The slight differences seen in the epitope stabilization assay are certainly too subtle for the short MD runs reported here. They probably result from: (i) the absence of a side chain at position P1 of ligand 4; and (ii) a weaker binding contribution of the bulging P4–P8 part, for which higher atomic fluctuations (data not shown) and less nonbonded contacts (see the high solvent accessibility of the P5 and P8 residues for ligand 4, Fig. 9) have been noticed. Energy-minimized timeaveraged conformations of both complexes (Figs. 11 and 12) clearly depict significant differences in the MHC pocket A (Tyr59, Trp167, Tyr171), which deviates dramatically from the starting crystal coordinates for B*2705 only (rmsd values from all pocket A atoms of 1.5 and 2.5 Å for B*2703 and B*2705, respectively). The major conformational alterations upon Bal1 binding were observed for the Tyr59-Trp167-Tyr171 triad (rmsd values of 1.7 and 2.7 Å for B*2703 and B*2705, respectively)sl. As predicted, the Tyr59 side chain was shifted away from the peptide N-terminus and is now interacting via a water molecule with the β-alanine terminal ammonium (Fig. 12). In contrast, the β-amino acid can directly interact with the larger pocket A of B*2703 through three Hbonds to His59 (mutated position), Glu63 and Glu163 (Fig. 11). Another significant difference in the binding of peptide 4 to both alleles concerns the C-terminal amino acid, which has nearly lost, upon binding to B*2705, all Hbonds to the polar side chains of pocket F (Tyr84, Thr143, Lys146; compare Figs. 11 and 12). The incorporation of a β-amino acid at P1 has modified the above described Hbond network between MHC side chains and the peptide N-terminus. The bound water molecule located in pocket A of HLA-B*2705 (recall Fig. 6) has either disappeared (B*2703, Fig. 11) or has been shifted towards the extreme left end of the binding groove (B*2705, Fig. 12). More importantly, these results show that the incor-

477 poration of a β-amino acid in the peptide sequence does not abrogate binding to HLA-B27 subtypes. Peptide 4 is one of the very first ligands for which the backbone modification of an anchor residue does not abolish class I MHC binding. Up to now, only a retro-inverso (NHCO instead of CO-NH) and a reduced peptide bond (CH2-NH instead of CO-NH) pseudopeptide analogue of an HLA-A2-binding peptide have been proposed as successful P1 modifications [64,65]. However, a β-amino acid at P1 presents the advantage to preserve the backbone direction of the peptide ligand and the H-bonding capacity of the first peptide bond (to Glu63 and Tyr159), so that less 3D conformational changes of the MHC binding cleft are necessary to accommodate the modified ligand. The recently described X-ray structure of an MHC–peptide– TcR ternary complex [66] suggests that the latter feature may be of particular importance for a proper recognition of the MHC–ligand pair by a TcR. Furthermore, it opens the door to the incorporation of β-amino acids at other anchor positions, notably P2, P3 and P9. Potential TcRbinding amino acids have already been replaced by various organic spacers without affecting the binding of the corresponding ligand to class I MHC proteins [38,67,68]. The present design study demonstrates that substituting a β-amino acid for a natural residue is a further solution for designing high-affinity MHC ligands with improved stability and pharmacokinetic properties. This is an absolute prerequisite for the therapeutic use of MHC ligands either as MHC blockers [69] or as T-cell receptor antagonists [70].

Conclusions MD simulations have been used in the present study as a tool for explaining peculiar structure–activity relationships at the level of the protein–ligand interaction complexes. The current study is not aimed at quantitatively ranking MHC ligands and predicting their binding affinities. For that purpose, free energy calculations using much longer equilibration and conformational sampling would be necessary. More simply, dynamical properties of the modeled complexes can be qualitatively well related to known binding data. Notably, monitoring protein– ligand intramolecular distances, the atomic mobility of the bound ligands, the ratio of accessible versus buried surface areas, the history and the quality of peptide– protein H-bonds allow a clear discrimination of highaffinity from weak-binding peptides. This computational approach, based on the qualitative analysis of short MD trajectories, has already been used to succesfully predict the bound conformation of a natural HLA-A2-restricted epitope [37] prior to X-ray structure determination [55], to identify T-cell epitopes from the primary structure of potentially interesting proteins [9] and to design highaffinity nonnatural ligands [38,71]. Herewith, we propose

its application to the rationalization of peptide specificity for closely related HLA alleles and the design of nonnatural ligands with increased specificity for one HLAB27 subtype. Identifying the molecular rules, fine tuning peptide selection by HLA alleles is a crucial step for better understanding the peptide–HLA interactions that may confer either susceptibility or resistance to immunological diseases associated with particular HLA alleles.

Acknowledgments D.R. wishes to thank the computational center of the ETH Zürich for generous allocation of computer time on the CRAY J90 and PARAGON machines. This work was supported by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung (Project No. 31-45504.95) and by Grant SAF 94-0891 from the Plan Nacional de I+D to J.A.L.C. J.R.L. is a fellow of the Basque Government.

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47 Berendsen, H.J.C., Postma, J.P.M., van Gunsteren, W.F., DiNola, A. and Haak, J.R., J. Chem. Phys., 81 (1984) 3684. 48 Klapper, I., Hagstrom, R., Fine, R., Sharp, K. and Honig, B., Proteins Struct. Funct. Genet., (1986) 47. 49 Warwicker, J. and Watson, H.C., J. Mol. Biol., 157 (1982) 671. 50 Gilson, M.K. and Honig, B., Proteins Struct. Funct. Genet., 4 (1988) 7. 51 Gilson, M.K., Sharp, K. and Honig, B., J. Comput. Chem., 9 (1988) 327. 52 Ljunggren, H.G., Stam, N.J., Ohlen, C., Neefjes, J.J., Hoglund, P., Heemels, M.T., Bastin, J., Schumacher, T.N., Townsend, A., Karre, K. and Ploegh, H.L., Nature, 46 (1990) 476. 53 Ellis, S.A., Taylor, C. and McMichael, A., Hum. Immunol., 5 (1982) 49. 54 van Gunsteren, W.F. and Berendsen, H.J.C., Angew. Chem., Int. Ed. Engl., 29 (1990) 992. 55 Madden, D.R., Garboczi, D.N. and Wiley, D.C., Cell, 75 (1993) 693. 56 Reid, S.W., McAdam, S., Smith, K.J., Klenerman, P., O’Callaghan, C.A., Harlos, K., Jakobsen, B.K., McMichael, A.J., Bell, J.I., Stuart, D.I. and Jones, E.Y., J. Exp. Med., 184 (1996) 2279. 57 Rovero, P., Vigano, S., Pegorado, S., Revoltella, R., Riganelli, D., Fruci, D., Greco, G., Butler, R. and Tanigaki, N., J. Pept. Sci., 1 (1995) 266. 58 Brunne, R.M., Berndt, K.D., Güntert, P., Wüthrich, K. and van Gunsteren, W.F., Proteins Struct. Funct. Genet., 23 (1995) 49. 59 Fox, T. and Kollman, P.A., Proteins Struct. Funct. Genet., 25 (1996) 315. 60 Gallego, J., Ortiz, A.R., de Pascual-Teresa, B. and Gago, F., J. Comput.-Aided Mol. Design, 11 (1997) 114. 61 Taylor, N.R. and von Itzstein, M., J. Comput.-Aided Mol. Design, 10 (1996) 233. 62 Bash, P.A., Singh, U.C., Langridge, R. and Kollman, P.A., Science, 236 (1987) 564. 63 Garcia, F., Marina, A., Albar, J.P. and López de Castro, J.A., Tissue Antigens, 49 (1997) 23. 64 Guichard, G., Calbo, S., Muller, S., Kourilsky, P., Briand, J.-P. and Abastado, J.-P., J. Biol. Chem., 270 (1995) 26057. 65 Guichard, G., Connan, F., Graff, R., Ostankovitch, M., Muller, S., Guillet, J.-G., Choppin, F. and Briand, J.-P., J. Med. Chem., 39 (1996) 2030. 66 Garboczi, D.N., Ghosh, P., Utz, U., Fan, Q.R., Biddison, W.E. and Wiley, D.C., Nature, 384 (1996) 134. 67 Weiss, G.A., Collins, E.J., Garboczi, D.N., Wiley, D.C. and Schreiber, S.L., Chem. Biol., 2 (1995) 401. 68 Bouvier, M. and Wiley, D.C., Proc. Natl. Acad. Sci. USA, 93 (1996) 4583. 69 Adorini, L., Muller, S., Cardinaux, F., Lehmann, P.V., Falcioni, F. and Nagy, Z.A., Nature, 334 (1988) 623. 70 De Magistris, M.T., Alexander, J., Coggeshall, M., Altman, A., Gaeta, F.C.A., Grey, H.M. and Sette, A., Cell, 68 (1992) 625. 71 Rognan, D., Habilitationsschrift, ETH Zürich, Zürich, Switzerland, 1997. 72 Saper, M.A., Bjorkman, P.J. and Wiley, D.C., J. Mol. Biol., 219 (1991) 277. 74 Kraulis, P.J., J. Appl. Crystallogr., 24 (1991) 846. 75 Merritt, E.A. and Murphy, M.E.P., Acta Crystallogr., D50 (1994) 869. 76 Connolly, M.J., J. Appl. Crystallogr., 16 (1983) 548.

ANEXO -4-

ANEXO -5-

ARTHRITIS & RHEUMATISM Vol. 42, No. 9, September 1999, pp 1975–1985 © 1999, American College of Rheumatology

1975

MODULATION AT MULTIPLE ANCHOR POSITIONS OF THE PEPTIDE SPECIFICITY OF HLA–B27 SUBTYPES DIFFERENTIALLY ASSOCIATED WITH ANKYLOSING SPONDYLITIS ´ R. LAMAS, ALBERTO PARADELA, FERNANDO RONCAL, and JOSE ´ A. LO ´ PEZ JOSE Objective. To investigate the rules governing peptide binding to HLA–B*2705, and to B*2704 and B*2706, which are 2 subtypes differentially associated with ankylosing spondylitis. Methods. Poly-Ala analogs carrying the HLA–B27 motif Arg-2, and substitutions at anchor positions P1, P3, or P⍀, were used to determine a binding score for each residue at each position. Binding was assessed in a quantitative epitope stabilization assay, where the cell surface expression of HLA–B27 was measured by flow cytometry as a function of peptide concentration. Results. Peptide anchor residues contributed additively to B*2705 binding. About 15% of the natural B*2705 ligands used a deficient P3 or P⍀ anchor, but never both, indicating that detrimental anchoring at one of these positions is always compensated by a good anchor at the other one. About 50% of the B*2705 ligands used suboptimal P1 residues. However, this was compensated with optimal P3 and/or P⍀ anchoring. Peptides that were longer than decamers used good anchor residues at the 3 positions, suggesting more stringent binding requirements. B*2704 and B*2706 differed in their residue specificity at P1, P3, and P⍀. The rules derived for B*2705 also applied to the known ligands of these 2 subtypes. Conclusion. The B*2705, B*2704, and B*2706 Supported by grant no. SAF97/0182 from the Plan Nacional de I⫹D, grant no. PM95-002 from the Spanish Ministry of Education, and an institutional grant from the Fundacio ´n Ramo ´n Areces to the Centro de Biologı´a Molecular Severo Ochoa. Dr. Lamas is a fellow of the Basque Government. Jose´ R. Lamas, Alberto Paradela, Jose´ A. Lo ´pez de Castro, PhD: Consejo Superior de Investigaciones Cientı´ficas, and Universidad Auto ´noma de Madrid, Madrid, Spain; Fernando Roncal: Pharmacia-Consejo Superior de Investigaciones Cientı´ficas, Centro Nacional de Biotecnologı´a, Madrid, Spain. Address reprint requests to Jose´ A. Lo ´pez de Castro, PhD, Centro de Biologı´a Molecular Severo Ochoa, Universidad Auto ´noma de Madrid, Facultad de Ciencias, Cantoblanco, 28049 Madrid, Spain. Submitted for publication February 22, 1999; accepted in revised form May 4, 1999.

DE

CASTRO

peptide repertoires are limited by the allowed residue combinations described in this study. The differential association of B*2704 and B*2706 with spondylarthropathy correlates with differences in their peptide specificity at multiple anchor positions. However, it is now possible to predict the peptide features that determine this differential binding to both subtypes. HLA class I proteins bind endogenous peptides and present them at the cell surface for recognition by cytotoxic T lymphocytes. These peptides, generally ranging in size from 8 to 12 amino acids but with a predominance of nonamers and decamers, bind to the class I molecule through interactions involving the peptide main chain, both peptide ends, and various peptide side chains. These interact in cavities or pockets of the peptide-binding site, which are formed by amino acid residues of the class I molecule. The structure of these pockets is modulated by HLA polymorphism, which affects the size and polarity of the pockets, and this determines the peptide-binding specificity of each class I allotype (1). Typically, the peptide repertoire bound to a given class I molecule shows limited diversity at some peptide positions (P) (2). These so-called main anchor residues interact with the HLA molecule and have a significant contribution to peptide affinity. However, only ⬃30% of the nonamers carrying the right main anchor residues will actually bind a given class I molecule (3). This is due to the significant influence of auxiliary anchor positions. Thus, the peptide specificity of an HLA class I molecule cannot be understood without knowing the suitability of different amino acid residues at these auxiliary positions. This is also essential for the prediction of putative ligands. The peptide-binding specificity of HLA–B27 has received much attention because of the strong association of this molecule with ankylosing spondylitis (AS)

1976

and reactive arthritis (4–6). Among other mechanisms, it has been proposed that an autoimmune response triggered by bacterial infections against a self peptide presented by HLA–B27 could be a primary pathogenetic event (7). This hypothesis is supported by recent studies in transgenic rats (8), and by the differential association of HLA–B27 subtypes with AS. Multiple HLA–B27 subtypes, including those that are predominant in whites (B*2705) and Asians (B*2704), are associated with this disease (9). However, B*2709 (10) and B*2706 (11–13) are less or not associated with AS. Thus, the peptide specificity of HLA–B27 and its alteration by subtype polymorphism becomes highly relevant to understand the differential association of closely related subtypes with AS. Presumably, an arthritogenic peptide should be selectively presented by the various disease-associated subtypes to autoimmune T cells that are putatively involved in the pathogenesis of spondylarthropathies. The main anchor positions of HLA–B27–bound peptides are P2 and the C-terminal residue (P⍀). Auxiliary anchor positions include P1, P3, and P7 (14,15). Arg-2 is the main anchor motif for all HLA–B27 subtypes, and the overwhelming majority of natural HLA– B27 ligands have this residue. C-terminal residues are more variable, being basic, aliphatic, and aromatic in B*2705, B*2703, and B*2710 (14,16–19), and aliphatic/ aromatic with subtype variability in B*2701, B*2702, B*2704, B*2706, B*2707, and B*2709 (16,20–23). Sequence studies of subtype-bound peptide repertoires have revealed the main anchor motifs and the residues occurring naturally at other peptide positions, but provide little information about the relative suitability of residues at a given position. Very few studies, all limited to B*2705 and B*2703 (18,24–27), have addressed the role of auxiliary anchor residues in determining the peptide specificity of HLA–B27. In this study, we have systematically explored the specificity of B*2705, B*2704, and B*2706 for P1, P3, and P⍀ residues using series of poly-Ala nonamer analogs carrying Arg-2. The following issues were addressed. First, we evaluated the relative suitability of residues at each of the 3 positions for binding to B*2705. This allowed us to rank the contribution of different residues at the various positions, providing a novel understanding of the peptide specificity of the “prototype” HLA–B27 molecule. Second, we demonstrated that binding of a given ligand to B*2705 is usually an additive function of the contribution of the different anchor residues, implying that interactive effects among amino acid residues at different peptide positions do not play, in general, a significant role. This allows a reliable

LAMAS ET AL

prediction of putative HLA–B27 ligands. Third, we demonstrated that B*2704 and B*2706 differ in their residue specificity at the 3 positions, P1, P3, and P⍀. This provides a novel understanding of the functional differences of the 2 subtypes that are differentially associated with AS. MATERIALS AND METHODS Synthetic peptides. Peptides were synthesized in an AMS 422 Multiple Peptide Synthesizer (Abimed, Langelfeld, Germany) using Fmoc chemistry, and purified by reversephase high-performance liquid chromatography. The correct composition and quantitation of the peptides was determined by amino acid analysis, as previously described (28). Peptides were stored as stock solutions at 4°C in water. Cell lines and monoclonal antibodies (mAb). RMA-S is a mutant cell line of the Rauscher virus–induced murine T cell lymphoma RBL-5 (H-2b), which has impaired transporterassociated antigen-processing (TAP)–mediated peptide transport (29,30) and low surface expression of class I major histocompatibility complex antigens that can be induced at 26°C (31). RMA-S transfectant cells expressing B*2705, B*2704, or B*2706 plus human ␤2-microglobulin have been previously described (32,33). The expression levels of B*2705 and B*2706 at 26°C were the same, and that of B*2704 was ⬃30% lower. These transfectants were grown in RPMI 1640 medium containing 25 mM HEPES buffer and 10% fetal calf serum (FCS; all from Life Technologies, Paisley, UK). HMy2.C1R (C1R) is a human lymphoblastoid cell line that has low expression of its endogenous class I antigens. Transfectants of these cells, with high expression of B*2705, were cultured in Dulbecco’s modified Eagle’s medium with 7.5% heat-inactivated FCS. The mAb ME1 (␣-HLA–B27 ⫹ B7 ⫹ B22) (34) was used as undiluted culture supernatant. Peptide-binding assay. The quantitative epitope stabilization assay used has been previously described (32). Briefly, RMA-S transfectant cells were incubated at 26°C for 24 hours in 96-well plates in RPMI 1640, 25 mM HEPES buffer, and 10% FCS. After incubation, plates were washed with sterile phosphate buffered saline, and peptides diluted in RPMI/ HEPES medium without FCS were added at a final concentration ranging from 10–4 to 10–9M. Cells were then incubated for 1 hour at 26°C, transferred at 37°C, and collected for flow cytometric analysis after 4 hours. This time point was chosen because there was a significant difference between the HLA– B27–associated fluorescence in the presence of a peptide ligand relative to its absence. In addition, peptides differing in their dissociation rates are more easily distinguished at this time point. For B*2704, whose peptide-induced stabilization after this time was too low, cells were collected after 2 hours at 37°C. At this time point, the difference between the HLA– B27–associated fluorescence in the presence or in the absence of a suitable ligand was comparable with that for B*2705 or B*2706 at 4 hours. HLA–B27–associated fluorescence on RMA-S transfectant cells was plotted as a function of peptide concentration. Binding was calculated as follows. First, a natural HLA–B27 ligand (RRYQKSTEL) was chosen as reference, and its molar

PEPTIDE SPECIFICITY OF B*2705, B*2704, AND B*2706

1977

concentration at 50% of the maximum fluorescence obtained with that peptide (C50) was calculated. Second, the molar concentration of each other peptide required to obtain the fluorescence value at the C50 of the reference peptide was found by interpolation, and this was designated as the EC50. EC50 values of ⱕ5 ␮M indicated high affinity, since these values were generally obtained with natural HLA–B27 ligands. EC50 values ⬎5 ␮M and ⬍50 ␮M were considered to reflect intermediate affinity. EC50 values ⱖ50 ␮M indicated low affinity. Peptides with an EC50 ⬎100 ␮M were considered not to bind, since their affinity was below the detection limits of this assay (32). The binding-promoting effect of substitutions at P1, P3, or P9 was calculated as the binding of the corresponding poly-Ala peptide carrying each substitution plus Arg-2, relative to the ARAAAAAAA (ARA7) peptide. This was expressed as the ratio between the EC50 of ARA7 and that of each analog. All calculations were carried out with the Origin program (MicroCal Software, Northampton, MA). Flow cytometric analysis was carried out as previously described (28). Isolation and sequencing of B*2705-bound peptides. Natural B*2705 ligands were isolated by acid extraction of immunoaffinity-purified B*2705 from C1R transfectant cell lysates as previously described (35). Peptides were sequenced by quadrupole ion trap mass spectrometry using a nanospray interface as described elsewhere (36). Assignment of residues with the same mass (for example, I/L, Q/K) was done on the basis of unambiguous matching with known human sequences in the protein database.

RESULTS Specificity of B*2705 for peptide anchor residues. The role of the P1, P2, and P⍀ anchor positions in binding to B*2705 was analyzed with 3 series of poly-Ala nonamers carrying Arg-2 and most of the amino acid residues at P1 or P3. A more restricted series, mainly including basic, aliphatic, and aromatic residues, was used for P⍀ (P9). The contribution of each residue was measured as the ratio between the EC50 of the corresponding poly-Ala analog and that of ARA7. This peptide bound to B*2705 with an EC50 of 30 ␮M, reflecting the contribution of the peptide main chain, Arg-2, and Ala side chains. P1. The most favored residue at position P1 was Arg, so that the RRA7 peptide bound ⬃3-fold better than ARA7 (Figure 1). H and aromatic (Y, W, F) residues were roughly equivalent to Ala (relative binding 0.7–1.5). A few residues, including K, G, I, and M, were slightly less favored than Ala (relative binding 0.6). Finally, acidic (D, E), polar (S, T, N, Q), and some aliphatic (L, V) residues were detrimental (relative binding ⱕ0.4). P3. The binding efficiency of P3 poly-Ala analogs spanned a wide range (Figure 1). The most favored

Figure 1. Relative binding of poly-Ala peptide analogs to B*2705. Each analog (x-axis) is represented by the 1-letter code of the amino acid residue introduced at P1, P3, or P9 into the ARAAAAAAA (ARA7) sequence. The reference peptide RRYQKSTEL (EC50 of 3 ␮M) is designated by an asterisk. Binding of each analog was measured as described in Materials and Methods and its relative binding (y-axis, logarithmic scale) was expressed as the ratio between the EC50 of ARA7 (30 ␮M) and that of the analog. Since the maximum amount of peptide tested was 100 ␮M, relative binding lower than 0.3, which indicates lack of binding in this assay (EC50 ⬎100 ␮M), could not be measured. For representation purposes only, a value of 0.25 was assigned to these analogs. Data are the mean of at least 2 independent experiments.

residue was W (relative binding 6), followed by F and some aliphatic (L, M) residues (relative binding 3 or higher). Other aliphatic (V, I), some polar (N, S), H, and Y residues were similar to Ala (relative binding 0.7–1.5). A number of chemically diverse residues, including T and especially R, and acidic (D, E), Q, G, and P residues were detrimental.

1978

LAMAS ET AL

Table 1. Natural ligands of B*2705 and binding scores of their P1, P3, and P⍀ residues* Ligand Octamers RRFFPYYV RRFTRPEH Nonamers ARLQTALLV RRYQKSTEL SRTPYHVNL RRLPIFSRL GRHGVFLEL RRIYDLIEL RRYPDAVYL GRFGSGMNM GRTFIQPNM LRFQSSAVM RRSKEITVR FRYNGLIHR KRFEGLTQR HRAQVIYTR SRYWAIRTR RRFMPYYVY SRVKLILEY RRFFPYYVY RRVLVQVSY RRISGVDRY RRIKEIVKK RRVKEVVKK ARLFGIRAK GRIDKPILK GRFEGTSTK GRAFVTIGK

Binding score

Ref. number

1-1----1 1-1----X

35 14

3-1-----1 1-2-----1 4-4-----1 1-1-----1 4-2-----1 1-2-----1 1-2-----1 4-1-----1 4-4-----1 5-1-----1 1-3-----1 3-2-----1 4-1-----1 3-3-----1 4-2-----1 1-1-----1 4-2-----1 1-1-----1 1-2-----1 1-2-----1 1-2-----1 1-2-----1 3-1-----1 4-2-----1 4-1-----1 4-3-----1

This study† 14 This study 16 This study 44‡ 45‡ 23 23 23 14 14 14 23 14‡ 16 This study 17,35 23 14,16§ 14 14 14,16 14 23 HIV‡

Ligand IRLRPGGKK RRWLPAGDA GRLTKHTKF KRFKEANNF RRFGDKLNF KRFSFKKSF TRYPILAGH KRVVINKDT Decamers KRFEETGQEL NRFAGFGIGL RRQDILDLWI GRFNGQFKTY RRYDRKQSGY GRWPGSSLYY GRKTGQAPGY GRILSGVVTK RKGGNNKLIK LRDNIQGITK KRWIILGLNK RRFVNVVPTF KRWQAIYKQF RRIKEIVKKH Undecamers RRYLENGKETL RRMGPPVGGHR WRLGSSDILNY Dodecamers RRFVNVVPTFGK

Binding score

Ref. number

4-1-----1 1-1-----3 4-1-----4 4-1-----4 1-1-----4 4-1-----4 5-2-----X 4-2-----X

HIV‡ 14 16,17 This study 16 16 16 46‡

4-1------1 5-1------1 1-5------1 4-1------1 1-2------1 4-1------1 4-X------1 4-2------1 1-5------1 5-5------1 4-1------1 1-1------4 4-1------4 1-2------X

This study This study HIV‡ 17 23 23 23 23 23 This study HIV‡¶ This study 23 16

1-2-------1 1-1-------1 2-1-------1

17 16 This study

1-1--------1

23

* Except for the peptides of viral or bacterial origin, all other peptides are from endogenous proteins of the cell. Residues that were scored are shown in boldface type in the sequences. The binding scores are rated 1–5, from high to low, on the basis of the binding efficiency of the corresponding poly-Ala analogs, as follows: 1 ⫽ EC50 ⱕ10 ␮M; 2 ⫽ EC50 11–20 ␮M; 3 ⫽ EC50 21–40 ␮M; 4 ⫽ EC50 41–80 ␮M; 5 ⫽ EC50 ⬎80 ␮M. X ⫽ score not determined. The reference numbers for previously reported ligands are given. Human immunodeficiency virus (HIV)–derived peptides were obtained from the HIV Molecular Immunology Database of Los Angeles National Laboratory (http://hiv-web.lanl.gov/). † Previously reported as an octamer (ARLQTALL) based on Edman sequencing (see ref. 16). Also found as a nonamer (ARLQTALLV) in B*2709 (see ref. 23). ‡ Peptide of viral or bacterial origin. § Reported as a nonamer also in B*2701 (see ref. 20) and as a decamer (RRISGVDRYY) in B*2703 (see ref. 17) and B*2710 (see ref. 19). ¶ A natural variant of this peptide with the L6M change is also known.

P9. Since P9 is usually a basic or nonpolar residue among B*2705-bound peptides (Table 1), the analysis of the contribution of this position to B*2705 binding was restricted to these residues. Pro was also tested, since it is a C-terminal motif among HLA–B73–bound peptides which, as in HLA–B27, also have Arg-2 (37). As expected, K, R, aliphatic, and Y residues were strongly favored. In contrast, F and, even more, W and Pro were disfavored (Figure 1). Additive contribution of anchor residues to peptide binding. In the next set of experiments, we analyzed whether binding of a natural B*2705 ligand could be explained by the additive contribution of anchor residues or whether this binding was a more complex function involving interactive effects. Thus, we tested the

binding of 2 natural ligands of B*2705 and of a series of poly-Ala analogs carrying 1 or more of the anchor residues of each ligand. We first measured how much the introduction of these anchor residues increased or decreased binding relative to ARA7. We then calculated the ratio between the relative binding of each ligand, or analog carrying multiple anchor residues of that ligand, and the additive value of the relative binding of poly-Ala analogs carrying single anchor residues. If the contribution of individual residues is additive, this ratio should be 1. If interactive effects among anchor residues play a role, this ratio should deviate significantly from 1. Two natural B*2705 ligands and their corresponding analogs were tested: RRYQKSTEL and KRYKSIVKY. The first one was used as the reference peptide to calculate

PEPTIDE SPECIFICITY OF B*2705, B*2704, AND B*2706

the EC50 for all other peptides. To account for experimental error in the determination of EC50 values, ratios between 1.5 (1.5:1) and 0.67 (1:1.5) were considered to reflect the additive contribution of anchor residues. This range was chosen because it was similar to the differences observed among EC50 values obtained in individual experiments when binding of a given peptide to B*2705 was repeatedly measured. As shown in Figure 2A, binding of most of the peptide analogs carrying multiple anchor residues of RRYQKSTEL (P1, P3, P7, P9) was accounted for, in 4 of 5 cases, by the additive contribution of the corresponding analogs carrying single anchor residues. Similarly (Figure 2B), binding of KRYKSIVKY analogs was accounted for, in 4 of 5 cases, by the additive contribution of analogs carrying single or a smaller number of anchor residues, with 1 case (KRAAAAAAY, ratio 0.6) showing a small deviation from the 0.67–1.5 range. These results indicate that, in general, the binding efficiency of a given peptide is a simple additive function of the contribution of individual anchor residues. However, mutual effects among peptide side chains may occasionally affect binding. The joint contribution of the P4–P6 and P8 residues was inferred from the ratio between the relative binding of each peptide and of its corresponding analog carrying the P1, P2, P3, P7, and P9 residues. Thus, the ratio between the binding of RRYQKSTEL relative to ARA7 (10-fold) and that of the RRYAAATAL analog (15-fold) was 0.67, indicating little contribution of P4–P6 and P8 to binding of the natural ligand (Figure 2A). In the second example (Figure 2B), the ratio between the binding of KRYKSIVKY relative to ARA7 (7.5-fold) and that of the KRYAAAVAY analog (4.3-fold) was 1.74, which is slightly outside the 0.67–1.5 range, and therefore compatible with some contribution of P4–P6 and P8 to binding of this natural ligand. Rules determining usage of anchor residues among natural HLA–B27 ligands. After observing the effect of individual residues at P1, P3, and P⍀ on binding and finding that their contribution was additive, it was possible to address the question of whether natural B*2705 ligands used only suitable anchor residues or used detrimental ones at these 3 positions. To examine this, we started with a database of 54 natural B*2705 ligands of known sequence, including 10 reported for the first time here, consisting of 2 octamers, 34 nonamers, 14 decamers, 3 undecamers, and 1 dodecamer (Table 1). The P1, P3, and P⍀ residue of each ligand was assigned a score ranging 1–5 according to the binding efficiency of the corresponding poly-Ala analog

1979

Figure 2. Relationship between binding of peptides carrying anchor residues of A, RRYQKSTEL or B, KRYKSIVKY. Solid bars indicate binding relative to ARAAAAAAA (ARA7), expressed as the molar ratio between the EC50 of ARA7 and that of each peptide (x-axis). For substitutions that were detrimental relative to A (T7, K1), the decrease on binding was expressed as 1 minus the relative binding. Open bars indicate the additive value of the relative binding of poly-Ala analogs carrying single substitutions at P1, P3, P7, or P9. The effect of V7 was not analyzed separately. Instead, the ARAAAAVAY analog was used. To account for experimental error in the determination of EC50 values, the data indicated by the solid and open bars were considered equal when their ratio was between 0.67 (1:1.5) and 1.5 (1.5:1). Ratio values outside this range are marked with an asterisk. Data are the mean of at least 2 independent experiments.

carrying this residue. Scores 1 and 2 were assigned to residues whose corresponding poly-Ala analogs bound better than ARA7 (EC50 30 ␮M). Score 1 was an EC50 of ⱕ10 ␮M, and score 2 was an EC50 of 11–20 ␮M. Score 3 was assigned to residues whose effect was similar to Ala (EC50 21–40 ␮M). Scores 4 and 5 corresponded to residues that were less suitable than Ala at the corre-

1980

sponding position (score 4 EC50 41–80 ␮M; score 5 EC50 ⬎80 ␮M). As shown in Table 1, 44 of the 51 peptides (86%) scored at P⍀ had optimal anchor residues (score 1), and only 7 (14%) had P⍀ residues similar or worse than Ala (scores 3 and 4). Similarly, good anchor residues were largely predominant at P3: 45 of 53 peptides (85%) were scored 1 (51%) or 2 (34%) at this position, and 8 peptides (15%) showed P3 residues with a score equal to or worse than that of Ala. In contrast, P1 residues with a score of 1 or 2 occurred in only 24 of 54 peptides (44%), and 26 (48%) had residues less suitable than Ala. This indicates that P1 is more permissive than P3 or P⍀. Two additional points are worth noting. First, the 11-mer and 12-mer peptides had good anchors (score 1 or 2) at the 3 positions. This suggests that the peptide repertoire that deviates from the canonical size of class I ligands (8–10 residues) has a more stringent residue specificity at P1, P3, and P⍀. Second, all of the peptides with P⍀ residues scoring 3 or higher (5 nonamers and 2 decamers) had an optimal (score 1) P3 residue, irrespective of P1. Conversely, P3 anchors scoring 3 or higher, which were observed in 5 nonamers and 3 decamers, always occurred with an optimal (score 1) P⍀ residue, also irrespective of P1. These results indicate that deficient anchoring at either P3 or P⍀ is always compensated with an optimal anchor at the other position. Finally, of the 30 peptides with P1 residues scoring 3 or higher, 18 (60%) had residues with a score of 1 or 2 at both P3 and P⍀, and all had this in at least 1 of these 2 positions. Seven peptides had detrimental (score 4 or 5) residues at P1 and at either P3 or P⍀, but in all these cases, the other position had an optimal (score 1) residue. Thus, detrimental P1 residues require at least an optimal P3 or P⍀ anchor. These rules derived for B*2705 also applied to the few known natural ligands of B*2704 and B*2706 (Table 2) and are likely to apply generally to other class I proteins. Modulation of P1, P3, and P⍀ specificity by B*2704 and B*2706 polymorphism. In these experiments, we addressed the effect of B*2704 and B*2706 polymorphism on residue selection at these 3 peptide positions. ARA7 bound to B*2704 and B*2706 more efficiently (EC50 10 ␮M and 9 ␮M, respectively) than to B*2705. This probably reflects a stronger interaction of Ala residues at 1 or more anchor positions. P1. The effect of the P1 residue on binding to B*2704 (Figure 3) was similar to that for B*2705, but with some differences. For instance, G rather than R was the most favored residue for B*2704, but its effect on

LAMAS ET AL

Table 2. Natural ligands of B*2704 and B*2706 and binding scores of their P1, P3, and P⍀ residues* Ligand B*2704 RRFFPYYV RRYQKSTEL RRIYDLIEL RRRWRRLTV GRLTKHTKF QRKKAYADF GRFNGQFKTY RRYLENGKETL B*2706 RRLRNHMAV IRHNKDRKV RRHWGGNVL RRYQKSTEL QRKKAYADF RRYLENGKETL

Binding score

Ref. number

1-1----1 1-1-----1 1-1-----1 1-2-----1 1-1-----4 3-X-----4 1-1------3 1-1-------1

36 21 44† 44† 21 21 21 47

1-1-----1 2-1-----1 1-1-----1 1-1-----1 1-X-----2 1-1-------1

21 21 21 21 21 47

* Except for peptides of viral origin, all other peptides are from endogenous proteins of the cell. Residues that were scored are shown in boldface type in the sequences. Scores were calculated on the basis of the binding efficiency of the corresponding poly-Ala analogs as described in the text. X ⫽ score not determined. Binding scores for B*2704 ligands are assigned as for B*2705 (see footnote to Table 1). Scores for B*2706 ligands were assigned as follows: 1 ⫽ EC50 ⱕ5 ␮M; 2 ⫽ EC50 6–10 ␮M; 3 ⫽ EC50 11–20 ␮M; 4 ⫽ EC50 21–40 ␮M; 5 ⫽ EC50 ⬎40 ␮M. † Peptide of viral origin.

increasing binding relative to Ala was smaller than the effect of R1 on B*2705. In addition, G and K were slightly detrimental for binding to B*2705 (Figure 1), but not for B*2704 binding. Otherwise, the pattern of P1 residues that were equivalent to Ala (H, and aromatic residues) or detrimental (acidic, polar, and aliphatic) was similar to that for B*2705. The effect of P1 substitutions on B*2706 binding (Figure 3) was different than that for B*2704, mainly in that only D was strongly detrimental, whereas E, polar (S, T, N), and aliphatic (V, I, L, M) residues were similar to Ala, and Q was favored, also in contrast to B*2705. These results indicate that HLA–B27 subtypes with an identical A pocket have nonidentical residue specificities. P3. B*2704 was similar to B*2705 in its acceptance of H and aliphatic (V, I, L, M) and aromatic (F, Y, W) residues at P3, and in that acidic (D, E), polar (S, T, N, Q), G, and P residues were disfavored (Figure 4). A difference was that, in contrast to B*2705 (Figure 1), Ala-3 was no worse than bulkier nonpolar residues for binding to B*2704. In addition, N was less suitable than Ala in B*2704 binding, but not in B*2705 binding. The specificity of B*2706 for P3 residues showed important differences in comparison with B*2704 (Fig-

PEPTIDE SPECIFICITY OF B*2705, B*2704, AND B*2706

1981

This subtype was similar to B*2705 in the detrimental effect of F, W, and P residues. B*2706 differed from B*2704 in that F was not detrimental relative to Ala or to other aromatic residues (Figure 5), and in its much stronger preference for bulky aliphatic residues than for Ala. As in B*2704, Y, W, P, and basic residues were detrimental. However, the detrimental effect of P was smaller on B*2706, and that of K was larger, in comparison with B*2704. DISCUSSION The strategy used in this study to analyze the peptide specificity of HLA–B27 was an epitope stabilization assay, which measures peptide binding to “empty” HLA–B27 molecules expressed on the surface of TAP-deficient cells. No in vitro binding assay fully reproduces peptide loading in vivo, since this is a highly organized and incompletely known process requiring physical association of the TAP transporter, several chaperones, and the HLA molecule. However, natural B*2705 ligands consistently bind with high affinity in our Figure 3. Relative binding of poly-Ala P1 analogs to B*2704 and B*2706. Each analog (x-axis) is represented by the 1-letter code of the amino acid residue introduced at P1 into the ARAAAAAAA (ARA7) sequence. The RRYQKSTEL peptide is designated by an asterisk. Binding of each analog was measured as described in Materials and Methods and its relative binding (y-axis, logarithmic scale) was expressed as the ratio between the EC50 of ARA7 (10 ␮M for B*2704 and 9 ␮M for B*2706) and that of the analog. Since the maximum amount of peptide tested was 100 ␮M, relative binding values lower than 0.1, which indicate lack of binding in this assay (EC50 ⬎100 ␮M), could not be measured. Data are the mean of at least 2 independent experiments.

ure 4). For instance, H, R, and polar residues were significantly favored relative to Ala in B*2706, and G, P, and acidic residues were less detrimental. In addition, nonpolar aliphatic and aromatic residues were more suitable than Ala, but Y was less favored than many other residues, including other aromatic ones. Ala itself was among the best residues in B*2704, but worse than many others in B*2706. These results indicate that B*2704 and B*2706 polymorphisms affect P3 specificity, so that these 2 subtypes differ from B*2705 and between each other in their residue preferences at this position. P9. B*2704 differed from B*2705 in the detrimental effect of basic and Y residues at P9 (Figure 5). In addition, although aliphatic residues were favored on B*2704, this was not significantly above the effect of Ala.

Figure 4. Relative binding of poly-Ala P3 analogs to B*2704 and B*2706. Each analog (x-axis) is represented by the 1-letter code of the amino acid residue introduced at P3 into the ARAAAAAAA (ARA7) sequence. The RRYQKSTEL peptide is designated by an asterisk. Peptide binding is expressed as described in Figure 3. Data are the mean of at least 2 independent experiments.

1982

Figure 5. Relative binding of poly-Ala P9 analogs to B*2704 and B*2706. Each analog (x-axis) is represented by the 1-letter code of the amino acid residue introduced at P9 into the ARAAAAAAA (ARA7) sequence. The RRYQKSTEL peptide is designated by an asterisk. Peptide binding is expressed as described in Figure 3. Data are the mean of at least 2 independent experiments.

assay (32), suggesting that the peptide specificity of HLA–B27 as measured in the present study closely reflects its specificity in vivo. Poly-Ala analogs are commonly used to compare the contribution of different amino acid residues at single positions, since the polyAla backbone provides a uniform background to which binding of peptide analogs can be related. Although the Ala side chain may have a contribution to binding at some peptide positions, its effects are minimized due to its small size and neutral chemical character. The data concerning peptide binding to B*2705 demonstrate that residue variability at P1, P3, or P⍀ can significantly affect binding. However, on the basis of our observations regarding the effect of the best residue at each of these positions on increasing binding relative to ARA7, the importance of these positions can be ranked as P9 ⬎ P3 ⬎ P1. Thus, K9 increased binding by a factor of 8, W3 by a factor of 6, and R1 by a factor of ⬃3. That multiple residues were detrimental emphasizes that peptide binding is determined by both positive and negative effects at individual positions. In particular, detrimental

LAMAS ET AL

residues can substantially reduce peptide affinity (1). Thus, the unsuitability of Pro-9 for binding to B*2705 is a critical difference in comparison with HLA–B73, which is an antigen that has the same B pocket as HLA–B27 and also binds peptides with Arg-2, but has Pro as a prominent C-terminal motif (37). Our study is consistent with a previous one (25) that also used poly-Ala peptide analogs to test the role of P3 and P9 on B*2705 binding. In particular, it confirms the suitability of basic and aliphatic residues at P9 and of nonpolar aromatic residues at P3. However, the reported suitability of F9 and relatively low acceptance of Y9 is in contrast to our results. This difference might be related to the fact that, in the previous study, a refolding assay was used to assess binding and the poly-Ala analogs used were different, since, besides R2, they had residues other than Ala at several anchor positions. The examination of residues present among natural HLA–B27 ligands reveals that the most suitable residues are predominant at P3 and P⍀. However, there is a significant allowance (⬃15%) for suboptimal residues at each of these positions, which is always compensated with the presence of an optimal residue at the other position. Because there are virtually no exceptions to this pattern among the known B*2705 ligands, this emerges as a significant constraint to the B*2705 peptide repertoire in applying it to the prediction of natural ligands, in addition to putative additional constraints imposed by proteasomal cleavage and peptide transport (38–41). That P1 was significantly more permissive for suboptimal residues among B*2705 ligands supports the smaller contribution of this position to peptide binding. However, this permissivity was limited by the requirement of a good anchor residue at P3 and/or P9. That these 3 positions generally contributed in an additive way to binding and, together with R2, restored much of the affinity of natural ligands, strongly suggests that a reliable prediction of HLA–B27 ligands can be done on the basis of P1, P3, and P⍀ only. In further support of this, nonpeptidic ligands with significant affinity for an HLA class I molecule can be obtained by keeping the P1–P3 and P⍀ residues of a given peptide ligand and substituting organic spacers for P4–P8 (42,43). Although the limited number of known ligands larger than 10-mers makes it somewhat risky to derive general rules, our data might suggest that peptides that are suboptimal in size (11-mers, 12-mers) bind in vivo only if they have good anchors at the 3 positions, P1, P3, and P⍀. A second issue addressed herein was the compar-

PEPTIDE SPECIFICITY OF B*2705, B*2704, AND B*2706

ison of the peptide specificities of B*2705 and B*2704. These 2 subtypes are both associated with AS (9) and differ in only 3 amino acid residues: D77S, V152E, and A211G. Aside from rather slight differences in residue specificity at P1 and P3, these 2 subtypes differ mainly at P⍀. The detrimental effect of basic P⍀ residues is consistent with the absence of these motifs among B*2704-bound peptides (21), and is consistent with findings in previous binding studies (32). In spite of the detrimental effect of C-terminal Y and F for binding to B*2704, these residues are C-terminal motifs for B*2704-bound peptides (21). However, as in B*2705, a detrimental C-terminal residue can occur among B*2704 ligands if they have optimal P3 residues. On the basis of our results, it is likely that the B*2705 and B*2704 peptide repertoires overlap to a significant extent. The overlapping repertoire probably consists mainly of peptides with C-terminal aliphatic residues, and peptides with C-terminal Y or F plus nonpolar P3 residues. The peptide-binding differences between B*2704 and B*2706 are particularly relevant because, in contrast to the former subtype, B*2706 is less or not associated with AS in various populations (11–13). B*2706 differs from B*2704 in 2 amino acid residues, H114D and D116Y, both of which are located in the ␤-pleated sheet floor of the peptide-binding site and can affect the interaction with at least the P3 and P⍀ residues of bound peptides. Previous sequencing studies revealed that a major difference between B*2706 and B*2704 was the absence of Y as a C-terminal motif in B*2706 (21). In vitro binding studies also have shown that aliphatic C-terminal residues were much better than Y for B*2706 binding, and that this difference was smaller for B*2704 binding (32). In agreement with these previous data, our results now confirm the high suitability of nonpolar aliphatic residues for B*2706 binding. In addition, the absence of Y as a C-terminal motif for this subtype is explained because bulky aliphatic and F residues bind more strongly, relative to Y, than in B*2704. Therefore, peptides with C-terminal Y will compete less advantageously in vivo for binding to B*2706. B*2704 and B*2706 showed additional differences in their P1 and P3 residue specificity, which have not been revealed by previous studies. Most noteworthy were the specificity differences at P3, especially those concerning the better acceptance of R and polar residues in B*2706, and the worse suitability of A and Y relative to many other residues. These differences are probably due mainly to the presence of D114 in B*2706, instead of H114 in B*2704 and other subtypes. P1 differences between B*2704 and B*2706 were surprising

1983

because both subtypes have an identical A pocket, which is the site where P1 binds. This suggests that long-range effects of distant polymorphic residues in HLA–B27 modulate its interaction with N-terminal peptide residues. Long-range effects on B pocket specificity have been previously observed in B*2701 (20). The different residue specificity of B*2704 and B*2706 at multiple anchor positions implies that their differential association with AS correlates not only with their differential acceptance of C-terminal Y, but also with a more complex modulation of their peptide repertoires, affecting at least also P1 and P3 residues. Therefore, B*2704 and B*2706 may differ significantly in their peptide repertoires, although they share common ligands (21). The actual extent of their overlap and the type of peptides that are bound in vivo to only 1 of these subtypes will require a more extensive analysis of their natural ligands. In conclusion, the results in this study revealed the preferences of B*2705 for P1, P3, and P⍀ residues and some major rules governing residue usage in these positions among natural ligands. This will allow a meaningful prediction of B*2705 ligands based on P1, P2, P3, and P⍀. Thus, from any protein putatively involved in disease-related T cell responses, it is now possible to select a rather limited number of peptides fulfilling these rules to test their role as HLA–B27–restricted antigens. In addition, our results show that 2 subtypes differentially associated with AS differ in their residue specificity at multiple anchor positions, suggesting that many peptides having R2 and C-terminal motifs common to B*2704 and B*2706 may nevertheless bind with different efficiency or to only 1 subtype. Since this point is crucial for assessing the nature of putative arthritogenic peptides, correlations between the binding score of different residues at each position and their actual usage among natural ligands should be determined, as was done for B*2705 in this study. However, this requires a more extensive database of natural B*2704 and B*2706 ligands, of which very few are yet known. This study has defined some major peptide features that shape HLA–B27–bound peptide repertoires, and has described how these features are modulated by disease-related subtype polymorphism. The peptidebinding specificity of HLA–B27 is probably a critical feature for its linkage to spondylarthropathy, but the pathogenetic role of this antigen is unlikely to be explained solely by its peptide-binding properties. The critical question as to which HLA–B27–bound peptides may become target antigens of autoimmune T cell responses in disease pathogenesis remains unanswered.

1984

LAMAS ET AL

ACKNOWLEDGMENTS We thank Jesu ´s Vazquez and Anabel Marina (Department of Protein Chemistry, CBMSO) for help in mass spectrometry, Juan Pablo Albar (Centro Nacional de Biotecnologı´a, Madrid, Spain) and Francisco Gavilanes (Universidad Complutense de Madrid) for help in peptide chemistry, and Manuel Ramos (CBMSO) for his contributions to the database of HLA–B27 ligands.

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susceptibility to spondylarthropathies. J Clin Invest 1996;98: 2764–70. Griffin TA, Yuan J, Friede T, Stevanovic S, Ariyoshi K, RowlandJones SL, et al. Naturally occurring A pocket polymorphism in HLA-B*2703 increases the dependence on an accessory anchor residue at P1 for optimal binding of nonamer peptides. J Immunol 1997;159:4887–97. Garcia F, Rognan D, Lamas JR, Marina A, Lopez de Castro JA. An HLA-B27 polymorphism (B*2710) that is critical for T-cell recognition has limited effects on peptide specificity. Tissue Antigens 1998;58:1–9. Garcia F, Galocha B, Villadangos JA, Lamas JR, Albar JP, Marina A, et al. HLA-B27 (B*2701) specificity for peptides lacking Arg2 is determined by polymorphism outside the B pocket. Tissue Antigens 1997;49:580–7. Garcia F, Marina A, Lopez de Castro JA. Lack of carboxylterminal tyrosine distinguishes the B*2706-bound peptide repertoire from those of B*2704 and other HLA-B27 subtypes associated to ankylosing spondylitis. Tissue Antigens 1997;49:215–21. Tieng V, Dulphy N, Boisge´rault F, Tamouza R, Charron D, Toubert A. HLA-B*2707 peptide motif: Tyr C-terminal anchor is not shared by all disease-associated subtypes. Immunogenetics 1997;47:103–5. Fiorillo MT, Meadows L, D’Amato M, Shabanowitz J, Hunt DF, Apella E, et al. Susceptibility to ankylosing spondylitis correlates with the C-terminal residue of peptides presented by various HLA-B27 subtypes. Eur J Immunol 1997;27:368–73. Rovero P, Riganelli D, Fruci D, Vigano S, Pegoraro S, Revoltella R, et al. The importance of secondary anchor residue motifs of HLA class I proteins: a chemometric approach. Mol Immunol 1994;31:549–54. Fruci D, Greco G, Vigneti E, Tanigaki N, Butler RH, Tosi R. The peptide-binding specificity of HLA-B27 subtype (B*2705) analyzed by the use of polyalanine model peptides. Hum Immunol 1994;41:34–8. Wen J, Wang J, Kuipers JG, Huang F, Williams KM, Raybourne RB, et al. Analysis of HLA-B*2705 peptide motif, using T2 cells and monoclonal antibody ME1. Immunogenetics 1994;39:444–6. Colbert RA, Rowland Jones SL, McMichael AJ, Frelinger JA. Differences in peptide presentation between B27 subtypes: the importance of the P1 side chain in maintaining high affinity peptide binding to B*2703. Immunity 1994;1:121–30. Villadangos JA, Galocha B, Garcia F, Albar JP, Lopez de Castro JA. Modulation of peptide binding by HLA-B27 polymorphism in pockets A and B, and peptide specificity of B*2703. Eur J Immunol 1995;25:2370–7. Ljunggren HG, Karre K. Host resistance directed selectively against H-2-deficient lymphoma variants: analysis of the mechanism. J Exp Med 1985;162:1745–59. Townsend A, Ohlen C, Bastin J, Ljunggren HG, Foster L, Karre K. Association of class I major histocompatibility heavy and light chains induced by viral peptides. Nature 1989;340:443–8. Ljunggren HG, Stam NJ, Ohlen C, Neefjes JJ, Hoglund P, Heemels MT, et al. Empty MHC class I molecules come out in the cold. Nature 1990;346:476–80. Galocha B, Lamas JR, Villadangos JA, Albar JP, Lopez de Castro JA. Binding of peptides naturally presented by HLA-B27 to the differentially disease-associated B*2704 and B*2706 subtypes, and to mutants mimicking their polymorphism. Tissue Antigens 1996; 48:509–18. Villadangos JA, Galocha B, Lopez de Castro JA. Unusual topology of an HLA-B27 allospecific T cell epitope lacking peptide specificity. J Immunol 1994;152:2317–23. Ellis SA, Taylor C, McMichael A. Recognition of HLA-B27 and related antigens by a monoclonal antibody. Hum Immunol 1982; 5:49–59. Paradela A, Garcia-Peydro M, Vazquez J, Rognan D, Lopez de

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36.

37. 38.

39. 40.

41.

Castro JA. The same natural ligand is involved in allorecognition of multiple HLA-B27 subtypes by a single T cell clone: role of peptide and the MHC molecule in alloreactivity. J Immunol 1998;161:5481–90. Yague J, Vazquez J, Lopez de Castro JA. A single amino acid change makes the peptide specificity of B*3910 unrelated to B*3901 and closer to a group of HLA-B proteins including the malaria-protecting allotype HLA-B53. Tissue Antigens 1998;52:416–21. Barber LD, Percival L, Parham P. Characterization of the peptidebinding specificity of HLA-B*7301. Tissue Antigens 1996;47:472–7. Van Endert PM, Riganelli D, Greco G, Fleischhauer K, Sidney J, Sette A, et al. The peptide-binding motif for the human transporter associated with antigen processing. J Exp Med 1995;182: 1883–95. Uebel S, Kraas W, Kienle S, Wiesmuller KH, Jung G, Tampe R. Recognition principle of the TAP transporter disclosed by combinatorial peptide libraries. Proc Natl Acad Sci U S A 1997;94:8976–81. Daniel S, Brusic V, Caillat-Zucman S, Petrovsky N, Harrison L, Riganelli D, et al. Relationship between peptide selectivities of human transporters associated with antigen processing and HLA class I molecules. J Immunol 1998;161:617–24. Peh CA, Burrows SR, Barnden M, Khanna R, Cresswell P, Moss DJ, et al. HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading. Immunity 1998;8:531–42.

1985

42. Rognan D, Scapozza L, Folkers G, Daser A. Rational design of nonnatural peptides as high-affinity ligands for the HLA-B*2705 human leukocyte antigen. Proc Natl Acad Sci U S A 1995;92: 753–7. 43. Krebs S, Lamas JR, Poenaru S, Folkers G, Lopez de Castro JA, Seebach D, et al. Substituting nonpeptidic spacers for the T cell receptor-binding part of class I major histocompatibility complexbinding peptides. J Biol Chem 1998;273:19072–9. 44. Brooks JM, Murray RJ, Thomas WA, Kurilla MG, Rickinson AB. Different HLA-B27 subtypes present the same immunodominant Epstein-Barr virus peptide. J Exp Med 1993;178:879–87. 45. Van Binnendijk RS, Versteeg-van Oosten JP, Poelen MC, Brugghe HF, Hoogerhout P, Osterhaus AD, et al. Human HLA class Iand HLA class II-restricted cloned cytotoxic T lymphocytes identify a cluster of epitopes on the measles virus fusion protein. J Virol 1993;67:2276–84. 46. Ugrinovic S, Mertz A, Wu P, Braun J, Sieper J. A single nonamer from the Yersinia 60Kd heat shock protein is the target of HLA-B27 restricted CTL response in Yersinia-induced reactive arthritis. J Immunol 1997;159:5715–23. 47. Garcia F, Marina A, Albar JP, Lopez de Castro JA. HLA-B27 presents a peptide from a polymorphic region of its own molecule with homology to proteins from arthritogenic bacteria. Tissue Antigens 1997;49:23–8.

ANEXO -6-

THE JOURNAL OF BIOLOGICAL CHEMISTRY © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 273, No. 30, Issue of July 24, pp. 19072–19079, 1998 Printed in U.S.A.

Substituting Nonpeptidic Spacers for the T Cell Receptor-binding Part of Class I Major Histocompatibility Complex-binding Peptides* (Received for publication, December 23, 1997, and in revised form, April 21, 1998)

Stefan Krebs‡, Jose´, R. Lamas§, Sorana Poenaru¶, Gerd Folkers‡, Jose´ A. Lo´pez de Castro§, Dieter Seebach¶, and Didier Rognan‡储 From the ‡Department of Pharmacy, Swiss Federal Institute of Technology, Winterthurerstrasse 190, CH-8057 Zu¨rich, Switzerland, the ¶Laboratory for Organic Chemistry, Swiss Federal Institute of Technology, Universita¨tstrasse 16, CH8092 Zu¨rich, Switzerland, and the §Centro de Biologia Molecular “Severo Ochoa,” Universidad Autonoma de Madrid, Facultad de Ciencas, E-28049 Madrid, Spain

X-ray diffraction studies as well as structure-activity relationships indicate that the central part of class I major histocompatibility complex (MHC)-binding nonapeptides represents the main interaction site for a T cell receptor. In order to rationally manipulate T cell epitopes, three nonpeptidic spacers have been designed from the x-ray structure of a MHC-peptide complex and substituted for the T cell receptor-binding part of several antigenic peptides. The binding of the modified epitopes to the human leukocyte antigen-B*2705 protein was studied by an in vitro stabilization assay, and the thermal stability of all complexes was examined by circular dichroism spectroscopy. Depending on their chemical nature and length, the introduced spacers may be classified into two categories. Monofunctional spacers (11-amino undecanoate, (R)-3-hydroxybutyrate trimer) simply link two anchoring peptide positions (P3 and P9) but loosely contact the MHC binding groove and thus decrease more or less the affinity of the altered epitopes to human leukocyte antigen-B*2705. A bifunctional spacer ((R)-3-hydroxybutyrate tetramer) not only bridges the two distant anchoring amino acids but also strongly interacts with the binding cleft and leads to a 5-fold increase in binding to the MHC protein. To our knowledge, this is the first report of a nonpeptidic modification of T-cell receptor binding residues that significantly enhances the binding of altered peptide ligands to their host MHC protein. The presented modified ligands constitute interesting tools for perturbing the T cell response to the parent antigenic peptide. Class I MHC1 molecules are highly polymorphic proteins that play a key role in immune surveillance by presenting foreign peptides to cytotoxic T lymphocytes (1). The molecular mechanisms of peptide selection have been characterized by x-ray diffraction studies of several MHC proteins in complex with either a peptide pool or single ligands (2). Peptides,

generally nonamers, tightly bind to conserved MHC residues in a sequence-independent manner at their N and C termini (3), whereas the central part of the bound peptide bulges out of the binding groove (4). Peptide specificity is governed by the position and chemical nature of some anchoring side chains (often P2, P3, and P9) that bind to MHC polymorphic pockets (5, 6). Complementary to x-ray structure determinations, sequencing self-peptides naturally bound to MHC proteins allows the determination of peptide binding motifs (7, 8) and thus the identification of conserved amino acids responsible for MHC binding (named dominant anchors, generally at positions P2 and P9) and more variable residues hypothesized to account for TcR recognition (usually in the central part of the peptide sequence, from P4 to P8). Peptide mutation (9, 10) as well as recently determined x-ray structures of ␣␤ TcRs in complex with a MHC-peptide (11, 12) unambiguously support this assumption. Since some class I MHC alleles are associated with either susceptibility or resistance to human diseases (13–15), altering TcR contact residues of T cell epitopes has been proposed for designing altered peptide ligands with TcR antagonist properties (16), leading to in vivo T cell anergy (17). However, natural peptides cannot be easily used as immunosuppressors because of poor enzymatic stability and pharmacokinetic properties (18). Herewith, we describe the substitution of nonpeptidic moieties for the TcR contact amino acids of several T cell epitopes naturally presented by the class I MHC protein B*2705, which is strongly linked to severe inflammatory diseases like ankylosing spondylitis (13) or reactive arthritis (19). Some reports in which a similar strategy has been followed (20 –22) show that the altered peptide ligands still form stable complexes with their host MHC protein but often present a reduced affinity relative to the parent peptide. The present study describes a novel oligomeric spacer able not only to link two MHC anchoring positions (P3 and P9) but also to significantly improve binding to the restricting class I MHC protein. EXPERIMENTAL PROCEDURES

* This work was supported by the Schweizerischer Nationalfonds zur Fo¨rderung der wissenschaftlichen Forschung (Project 31-45504.95); Ministry of Education Grant PM95– 002 and Plan Nacional de I ⫹ D Grant SAF97– 0182 (to J.A.L.C.); and an institutional grant of the Fundacion Ramon Areces to the Centro de Biologia Molecular “Severo Ochoa.” The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 储 To whom correspondence should be addressed. Fax: 41-1-635-68-84; E-mail: [email protected]. 1 The abbreviations used are: MHC, major histocompatibility complex; HLA, human leukocyte antigen; TcR, T cell receptor; Pn, peptide position n; Fmoc, N-(9-fluorenyl)methoxycarbonyl; Aua, 11-amino undecanoate; HB, (R)-3-hydroxybutyrate.

Computer-assisted Ligand Design—Molecular mechanics and dynamics calculations were carried out using the AMBER 4.1 package (23), using the parm94 parameter set (24) and an all-atom force field representation. Force field parameters for the ester group were taken from the literature (25). Atomic charges for the Aua and HB monomers were calculated using the GAUSSIAN 94 package (26) and the HF/6 – 31G* basis set by fitting atom-centered charges to an ab initio electrostatic potential, using the RESP method (27) according to a previously described procedure (28). Atomic charges for both new monomers are listed in Table I. Initial coordinates for the MHC-ligand complexes were obtained from the x-ray structure of HLA-B*2705 (3) as described previously (20, 29). The spacers were substituted for the natural pentapeptide sequence using the SYBYL modeling package (TRIPOS Association, Inc., St.

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Nonnatural HLA-B27 Ligands

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TABLE I Restrained electrostatic potential-derived point charges, calculated using the RESP method (26) from GAUSSIAN 94 HF/6 –31G* electrostatic potentials

a Ab initio derived electrostatic potentials have been calculated for protected monomers (Ac-Aua-NMe, Ac-HB-OMe) and atomic charges of the isolated monomers (Aua and HB) adjusted to neutrality using Lagrange constraints, as previously described (28). Charges for equivalent hydrogen atoms are only mentioned once. b 11-aminoundecanoate. c (R)-3-hydroxybutyrate.

Louis, MO). From a starting fully extended conformation, dihedral angles of the main chain between P3 and P9 were modified by hand in order to reproduce a correct trans geometry for the newly introduced amide or ester bonds. The ligand was first relaxed by 500 steps of conjugate gradient energy minimization while maintaining the protein fixed. It was then submitted to a 100-ps simulated annealing protocol in order to sample the broadest conformational space accessible. Starting with random velocities assigned at a temperature of 1000 K, the ligand was first coupled for 50 ps to a heat bath at 1000 K using a relatively weak temperature coupling constant ␶ (0.2 ps) and then linearly cooled down to 50 K for the next 50 ps while ␶ was strengthened to a value of 0.05 ps. During these 100 ps, no protein atoms were allowed to move. The last conformer was then solvated in a 10-Å-thick TIP3P water shell. Energy minimization of the ligand, of the MHC-ligand complex, followed by 200-ps molecular dynamics simulation of the fully solvated MHC-ligand pair was performed as previously reported (20). Synthesis of the Modified Peptides—Ligands 1– 8 (Table II) were obtained by automated solid-phase peptide synthesis using a Fmoc/tertbutyl protecting strategy. Chain elongation was performed by a robot system (Syro Multi-Syn-Tech, Bochum, Germany) with a subsequent manual deprotection and analysis. Fmoc-protected amino acids were coupled to the diisopropylcarbodiimide-activated carboxyl terminus in 10-fold excess using 1-hydroxybenzotriazole as a coupling reagent. The final peptide was simultaneously cleaved from the resin and deprotected by the addition of trifluoroacetic acid with thiocresole and thioanisole as scavengers. The peptides were precipitated and washed with ice-cold ether and further lyophilized from water. Natural as well as nonnatural peptides were analyzed by reverse phase high performance liquid chromatography (Merck-Hitachi, Darmstadt, Germany) on a nucleosil 5␮, C-18 column (125 ⫻ 3 mm) at a flow rate of 600 ␮l/min. Absorbance was measured at 220 nm. The solvent system consisted of 0.1% trifluoroacetic acid in water (buffer A) and 0.1% trifluoroacetic acid in acetonitrile (buffer B). A linear gradient from 10 to 60% B in 30 min was applied. Furthermore, peptides were analyzed by ion spray mass spectrometry on a triple quadrupole mass spectrometer, APII III, with a mass range of m/z 10 –2400 equipped with an ion spray interface (Sciex, Thornhill, Canada). The mass spectrometer was operated in positive ion mode under conditions of unit mass resolution for all determinations.

The synthesis of ligands 9 –12 will be reported elsewhere.2 Epitope Stabilization Assay—The quantitative assay used was described previously (30). Briefly, RMA-S transfectants expressing B*2705 or B*2704 were used. These are murine cells with impaired TAP-mediated peptide transport and low surface expression of (empty) class I MHC molecules, which can be induced at 26 °C (31) and stabilized at the cell surface through binding of exogenously added ligands. These cells were incubated at 26 °C for 24 h. After this, they were incubated 1 h at 26 °C with 10⫺4 to 10⫺9 M peptides, transferred to 37 °C, and collected for flow microcytometry analysis with the ME1 monoclonal antibody (IgG1, specific for HLA-B27, -B7, and -B22) (32) after 4 h for B*2705 or after 2 h for B*2704. The determinant recognized by ME1 is not affected by bound peptides or by polymorphism in these two subtypes (data not shown). Binding of a given ligand was measured as its C50. This is its molar concentration at 50% of the fluorescence obtained with that ligand at 10⫺4 M. Ligands with C50 ⱕ 5 ␮M were considered to bind with high affinity, since these were the values obtained for most of the natural B27-bound peptides. C50 values between 5 and 50 ␮M were considered to reflect intermediate affinity. C50 ⱖ 50 ␮M indicated low affinity. Binding of peptide analogs was measured as the concentration of the peptide analog required to obtain the fluorescence value at the C50 of the unchanged peptide. This was designated as EC50. Relative binding was the ratio between the EC50 of the peptide analog and the C50 of the corresponding unchanged peptide. HLA-B*2705 Expression and Purification—A cDNA encoding for human ␤2-microglobulin (gift of Dr. C. Vilches, Clinica Puerta de Hierro, Madrid) was cloned into a pGex vector (Amersham Pharmacia Biotech), yielding a fusion protein with glutathione S-transferase. Escherichia coli cells transformed with this pGex vector were grown under vigorous shaking in LB broth for 24 h at 25 °C after induction with isopropyl-1thio-␤-D-galactopyranoside. Cells were frozen at ⫺70 °C, thawed, suspended in TBS (20 mM Tris, 150 mM NaCl, pH 8.0), and lysed by the addition of lysozyme and brief sonication. The crude extract was passed over a glutathione-agarose column (Sigma), and after extensive wash-

2 D. Seebach, S. Poenaru, G. Folkers, and D. Rognan, manuscript in preparation.

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Nonnatural HLA-B27 Ligands TABLE II Binding of natural and altered T cell epitopes EC50a

Sequence Ligand P1

P2

P3

Spacer

P9

B*2705

R

R

R

Vc

S

R

Y

G

R

A

Q

R

L

WRRLT Auad WAIRT Aua FVTIG Aua KEAAE Aua (HB)3h (HB)4 (HB)3 (HB)4

1.2 4.0 3.0 8.6 1.8 7.0 10.0

B*2704

␮M

1 2 3 4 5 6 7 8 9 10 11 12

A

Re Kf Kg

Tmb °C

0.8 1.0 5.4 100.0 6.4 ⬎100.0

40.0 2.5 20.0 1.6

52.8 ⫾ 0.7 42.9 ⫾ 0.3 46.3 ⫾ 0.5 39.5 ⫾ 0.2 61.9 ⫾ 0.3 48.1 ⫾ 0.4 62.8 ⫾ 0.7 46.5 ⫾ 0.2 63.2 ⫾ 0.6 62.1 ⫾ 0.7

a Concentration of ligand at which HLA-B27 fluorescence (measured by flow microcytometry analysis with an anti-B27 monoclonal antibody) on RMA-S cells was half the maximum obtained with the wild type peptide (30). b Melting temperature: midpoint of the thermal unfolding of the B*2705 heavy chain. Tm values are means of three denaturation experiments performed on independently reconstituted heavy chain-␤2-microglobulin-ligand heterotrimers. S.D. values have been obtained by fitting the obtained curves to a two-state model as previously described (35). c Epstein-Barr virus latent membrane protein (236 –244) (49). d

e f g

h

Aua:

11-amino undecanoate.

Influenza A nucleoprotein (383–391) (50). Human immunodeficiency virus 1 glycoprotein 120 (314 –322) (39). E. coli DnaK protein (260 –268) (20). HB:

(R)-3-hydroxybutyrate.

ing with TBS, the ␤2-microglobulin was eluted by thrombin cleavage as a single band at 11 kDa (SDS-polyacrylamide gel electrophoresis). The HLA-B*2705 heavy chain was affinity-purified under denaturing conditions as a His6 fusion protein. The expression vector was obtained by subcloning the cDNA encoding for the first extracellular 274 amino acids (gift of Dr. K. C. Parker, National Institutes of Health, Bethesda) into the polycloning site of the oligohistidine vector pQE30 (Quiagen) with the restriction endonucleases BamHI and HindIII. The heavy chain was expressed in E. coli at 35 °C for 2 h after induction with isopropyl-1-thio-␤-D-galactopyranoside. Longer expression times led to an increase of immature or degraded heavy chains. Inclusion bodies were prepared using a standard procedure (33) and solubilized in 8 M urea, 20 mM Tris, 150 mM NaCl at pH 8.0. Purification on a nickel-nitriloacetate-agarose column led to the HLA-B*2705 heavy chain with two minor impurities of lower molecular weights consisting of truncated heavy chains. Folding of the MHC Protein upon Ligand Binding—Reconstitution of the heavy chain-␤2-microglobulin-ligand heterotrimer was achieved by dialysis (cellulose ester tubings, 500-Da cut-off) of a solution containing 0.15 mg/ml heavy chain, 0.1 mg/ml ␤2-microglobulin, and 0.1 mg/ml peptide ligand, using 5 mM glutathione to establish reducing conditions in 6 M urea against TBS. The solution was sparged with nitrogen to prevent premature formation of disulfide bridges and oxidation of free Cys67 in the B*2705 heavy chain. After 36 – 48 h at 10 °C, the mixture was concentrated to 500 ␮l in a Centripep ultrafiltration unit (AmiconGrace Ltd.). The folded heterotrimer was purified by gel filtration on a superdex 75 column (Amersham Pharmacia Biotech) with UV detection at 280 nm. The chromatogram showed three major peaks at 9-, 11.5-, and 14-ml elution volume corresponding to heavy chain aggregates, refolded complex, and excess ␤2-microglobulin, respectively. The overall yield of the fully reconstituted heterotrimer varied around 5%. The heterotrimer peak was collected, concentrated in a Centricon 30 ultrafiltration unit (Amicon-Grace), and immediately subjected to thermal denaturation. Monitoring the Thermal Stability of MHC-Ligand Complexes by CD Spectroscopy—All CD measurements were done on a Jasco J-720 polarimeter with a water-jacketed 1-mm sample cell connected to a computer-interfaced Neslab 111 circulating water bath. Temperature control was achieved by measuring the circulating water immediately after the sample cell. The thermal denaturation profiles were recorded at 218 nm in 10 mM Tris, 150 mM NaCl (pH 8.0) with the Jasco TEMPSCAN software using 0.1 °C increments at a heating rate of 30 °C/h. Sample concentrations were determined photometrically and held at 0.2 mg/ml. Different scan rates did not affect the Tm value of B*2705 in complex

with a reference peptide (GRAFVTIGK; compare Ref. 34 and Table II). Three denaturation curves from independent refolding preparations were averaged, after conversion to molar ellipticity values. The curves were reduced to 70 data points by replacing each of the 10 neighboring points with their mean value. By assuming a two-state equilibrium (35), data were fitted by a nonlinear least-squares routine with the program Origin 2.9 (MicroCal Software, Inc.) to the following equations. ⌰共T兲 ⫽ ⌰u ⫹ 共⌰f ⫺ ⌰u/1 ⫹ exp共x兲兲 x ⫽ 共⫺⌬Hm/R兲共1/T ⫺ 1/Tm兲 ⫹ 共⌬Cp/R兲共共Tm/T ⫺ 1兲 ⫹ ln共T/Tm兲兲

(Eq. 1) (Eq. 2)

The measured ellipticity (⌰) is given as a function of the temperature (T) with the enthalpy (⌬Hm), heat capacity upon unfolding (⌬Cp), and the midpoint temperature of unfolding (Tm) being the fitting parameters. Initial estimates for ⌬Hm were obtained by plotting ln K versus 1/T (van’t Hoff plot) in the transition region. ⌬Cp was assumed to be temperature-independent (36), and initial values were estimated from the primary sequence (37). The linear base-line functions of the unfolded and folded states ⌰u and ⌰f were determined as linear regressions of the pre- and post-transitional regions. The enthalpy change at the midpoint of unfolding (⌬Hm) was determined by the least-squares fit of the unfolding curve to Equation 1. Because ⌬Cp estimates obtained by this approach are not very accurate and the ⌬Hm values are largely influenced by the observed deviations from a two-state model, a direct extrapolation from the midpoint of unfolding to obtain ⌬⌬Gunfolding at 25 °C was not taken into consideration. RESULTS

Replacing a Pentapeptide with a Polymethylene Spacer in Four Unrelated Natural Epitopes—For mimicking the sequence of the central pentapeptide part (P4 –P8) of MHC-bound nonapeptides, any nonpeptidic fragment needs first to reproduce as closely as possible the conformation of this bulging part and second to allow the same intermolecular distance between the neighboring anchoring positions (P3 and P9) that are linked by the new spacer. The key distance between C-␣ atoms of P3 and P9 positions is 16.6 Å in the x-ray structure of HLA-B*2705 complexed by a nonapeptide model (3). The same distance can be easily obtained after linking a polymethylene chain (Aua, Fig. 1) to P3 and P9 residues by simple amide bonds. The 11-amino undecanoate fragment was then chosen

Nonnatural HLA-B27 Ligands

FIG. 1. Chemical structure of the modified peptide analogues.

for its optimal length in an extended conformation and the absence of any substituents, which should allow a conformational flexibility sufficient for a proper fit into the binding groove. To check the independence of the proposed modification on the parent epitope sequence, the Aua spacer was introduced in four unrelated sequences of natural epitopes, known to bind to B*2705 (Table II). The question of whether the new ligands were able to remain tightly bound in the peptide binding cleft like the natural nonapeptides was addressed by molecular dynamics simulations of the solvated complexes (Table II). The computational protocol used has been previously shown to explain the binding potency of several HLA-B27-binding peptides (38) and to predict the high affinity of designed peptide analogues (20, 29). Energy-minimized conformations show that the proposed bridging has modified neither the overall conformation of the bound ligands nor the intermolecular distance between P3 and P9 C-␣ atoms (Fig. 2). Moreover, the chemical substitutions were compatible with the conservation of the main interactions between the altered peptides and B*2705, especially the electrostatic interactions provided by the two charged termini and the arginine found at position 2 of B*2705binding peptides (39, 40). In order to experimentally validate the proposed model, the four B*2705-restricted T cell epitopes and their modified analogues (Table II) were synthesized and then tested for their binding to B*2705, in an in vitro epitope stabilization assay (30). Replacing the central pentapeptide sequence by the unsubstituted Aua fragment led in all cases to a slight decrease in B*2705 stabilization (Table II), which was also reflected by a lesser thermal stability of the resulting complexes monitored by CD spectroscopy (Fig. 3, A–D). The temperature shift in the midpoint of unfolding depends on the sequence of the reference peptide but varies from ⫺7 to ⫺14 °C (Table II). Whereas the effect of the Aua spacer is similar in both assays, there seems to be no clear correlation between the EC50 scores obtained from the epitope stabilization assay and the Tm values calculated from the thermal denaturation experiments. The Tm val-

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ues reported here are fairly similar to those found for B*2705binding peptides by other groups (21, 34, 41). The highest stability against temperature was found for complexes with peptides 5 and 7, which all present a lysine at P9. Less favored amino acids at P9 are Val (peptides 1 and 2) and especially Arg (peptides 3 and 4), which gives by far the least stable complexes with B*2705 (peptides 1 and 3, respectively). Binding of peptides 1– 6 to a closely related HLA-B27 subtype (B*2704) was also examined by the same in vitro stabilization assay. B*2704 differs from B*2705 by two amino acid changes in the peptide binding groove (Asp77 to Ser; Val152 to Glu), which influence its peptide specificity, relative to B*2705 (42). In contrast to B*2705, substitution of Aua spacers for P4 –P8 dramatically decreases binding to B*2704 in our epitope stabilization assay (Table II) when the last anchoring position (P9) is a basic amino acid (Lys, Arg). If P9 is an apolar residue (Val, peptide 2), no real change in B*2704 binding was noticed. Substituting 3-Hydroxybutyrate Oligomers for the P4 –P8 Sequence of a Natural Peptide—The decreased binding of the Aua analogues to B*2705 is probably due to the nonfunctionalized nature of the introduced spacer and the lack of interactions between the unsubstituted Aua moiety and the central part of the binding groove. Thus, a rational improvement in terms of binding affinity would be to ramify the spacing moiety in order to reach one of the two central pockets (pockets C and E) of the peptide binding groove that face the spacer fragment. The (R)-3-hydroxybutyrate (HB) monomer was selected for three main reasons: (i) polymers of HB are chemically stable (43); (ii) they adopt conformations whose folding in the free state resembles that found for peptides (44); and (iii) the methyl substituent is large enough to fit into pockets C and E. Thus, a trimer (three units) and a tetramer (four units) of HB were substituted for the P4 –P8 sequence of one natural peptide (polyesterpeptides 9 and 10; Table II), since they should optimally span the key distance between the two anchor positions (P3 and P9) to bridge (Fig. 1). In order to circumvent cyclization of the N-terminal glutamine (45) that would prevent binding of the peptidic N terminus in the A pocket of B*2705, the Ala1 analogue was also synthesized in the nonnatural series (polyesterpeptides 11 and 12; Table II). The modified ligands 9 –12 have totally different binding affinities in the in vitro stabilization assay (Table II), the tetramer-containing compounds (ligands 10 and 12) being about 15 times more potent that the trimeric analogues (ligands 9 and 11). Furthermore, a HB tetramer segment leads to a significant enhancement of the binding to B*2705 relative to the natural pentapeptide sequence. Again, the differences observed between natural and polyesterpeptides in the in vitro stabilization assay are not reflected by the thermal denaturation experiments, performed only for ligands 11 and 12. Both compounds promote a similarly high stability of the resulting MHC-ligand pair with Tm values of 62– 63 °C (Fig. 4, Table II) analogous to that found for the parent peptide 7, and characteristic of high affinity ligands (41). Molecular Modeling of the Altered Peptides in Complex with B*2705—A rationale for the (de)stabilizing effects of the three spacers presently studied is proposed by the molecular dynamics time-averaged conformations of a reference peptide (QRLKEAAEK; peptide 7) and its analogues (peptides 8 –10). By looking at all close nonbonded contacts between any peptide residue and its protein neighboring atoms, the three spacers (Aua, HB trimer, and HB tetramer) can be easily distinguished (Fig. 5). The Aua spacer provides fewer contacts to the MHC binding groove than the pentameric P4 –P8 sequence of the parent peptide 7. This could explain the decreased binding

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FIG. 2. Close-up into the binding groove of HLA-B*2705 (orange surface) in complex with peptides 7–10 (Table II). These structures represent energy-minimized conformations obtained from the x-ray structure of HLA-B*2705 in complex with a model peptide (Protein Data Bank entry 1hsa) under a previously described protocol (20, 29). The heavy chain backbone atoms of HLA-B*2705 have first been fitted together in the four complexes, and the protein atoms are not shown for the sake of clarity. Since protein distortion upon energy minimization of the resulting complexes is minimal, the MHC protein is here represented by a unique molecular surface independent of the bound ligand. The color coding is as follows: blue, nitrogen; red, oxygen; white, carbon atoms of ligand 7; cyan, carbon atoms of ligand 8; green, carbon atoms of ligand 9; yellow, carbon atoms of ligand 10. The arrows indicate two methyl substituents of the HB tetramer interacting with the central pockets C/E of the protein. The figure has been prepared using the program GRASP (51).

affinity of Aua-containing peptides to B*2705. The detrimental effect of the HB trimer can be explained by the weakening of the interactions between both terminal residues (PN and PC) and their respective pockets (A and F). The better complementarity of the HB tetramer to the B*2705 binding cleft is probably related to the following factors: (i) the additional interactions provided by two methyl groups of the tetrameric spacer itself and (ii) a higher number of nonbonded contacts of all other MHC anchors (PN, P2, P3, and PC). The total buried surface area of the modified ligands 8 –10 has been maintained when compared with that of the parent peptide 7 (about 650 Å2; data not shown). However, the total accessibility of the ligands in their bound state is different. It is reduced by 20% for HB analogues with respect to the natural epitope 7 (from 500 to 400 Å2). The Aua compound 8, although slightly less potent, has a much lower accessible surface area (250 Å2) due to the lack of substituents in the spacing area.

DISCUSSION

Replacing the central TcR-binding residues of MHC class I-bound peptides (P4 –P8) by nonpeptidic moieties has been reported previously (20, 21). Herewith, we propose to rationalize the effect of three novel spacers on binding to the HLAB*2705 protein. The simplest spacer (Aua) is a single polymethylene chain linking the P3 and P9 positions by amide bonds. In accordance with a previous report studying the effect of non-␣ amino acids (20), the Aua spacer does not impair binding to B*2705. Only a moderate decrease in relative binding to B*2705 was observed in an epitope stabilization assay, performed for four unrelated modified peptides (Table II). However, the effect of this modification on the thermal stability of the resulting MHC-ligand pair was more significant (Fig. 3, A–D). Depending on the peptide in which the Aua moiety was introduced, the midpoint of unfolding of the B*2705 heavy chain (Tm) was lowered by 7–14 °C. The corresponding free

Nonnatural HLA-B27 Ligands

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FIG. 3. Thermal denaturation, monitored by CD spectroscopy at 218 nm, of HLA-B*2705 loaded with ligands 1 and 2 (A), ligands 3 and 4 (B), ligands 5 and 6 (C), and ligands 7 and 8 (D). The arrows indicate the midpoint of unfolding (Tm) of the B*2705 heavy chain.

FIG. 4. Thermal denaturation, monitored by CD spectroscopy at 218 nm, of HLA-B*2705 loaded with ligands 7, 11, and 12.

energy change in unfolding ⌬⌬Gunfolding at the midpoint of unfolding, derived from the CD spectra (22), varies from ⫺0.8 to ⫺1.3 kcal/mol. Since unfolding of the heavy chain should follow release of the ligand, this observation supports a faster dissociation of the modified peptides with respect to the parent epitope, as recently illustrated in a homogeneous series of H-2Kd-binding nonapeptides (46). However, the present study suggests that extrapolating peptide binding differences from Tm values is not allowed for unrelated sequences. For the set of 4 T cell epitopes presently studied, EC50 values cannot be

FIG. 5. Nonbonded interactions between the HLA-B*2705 protein and ligands 7–10, measured on energy-minimized time-averaged conformations obtained after 200-ps Molecular Dynamics simulations of the corresponding solvated complexes. Protein-ligand contacts are recorded for interaction distances up to 4 Å.

related to melting temperatures calculated by CD spectroscopy. A likely explanation for this is that binding, as measured in epitope stabilization assays, is significantly influenced by the association rate of the peptide, whereas CD measurements relate only to the dissociation rates. The highest thermal stabilities were obtained for the B*2705 protein in complex with

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peptide ligands bearing a Lys at P9. This makes sense, since Lys is the P9 residue most complementary to its binding pocket F. Its side chain forms a buried salt bridge with Asp116, located at the bottom of the pocket. The predominance of the enthalpic contribution to peptide dissociation would thus be compatible with the lower Tm values observed with peptides having an amino acid (Val, Arg) for which the interaction with pocket F is weaker. It also corroborates previous computational simulations, suggesting that peptide dissociation first occurs at the C terminus (20, 29, 38). Interestingly, the effect of the Aua spacer is subtype-dependent, since differences between the natural and the Aua peptides in binding to B*2704 were much more significant (Table II). B*2704 basically differs from the B*2705 allele by its weak propensity to present peptides with basic P9 amino acids and its improved suitability for nonpolar P9 residues (42). Thus, the deleterious effect of the Aua spacer is amplified for peptides bearing a weak anchoring amino acid at P9 (peptides 4 and 6; Table II) and decreased for peptides with nonpolar P9 residues (peptide 2). The Aua group can be considered as a monofunctional spacer, since it simply provides the covalent linkage between two neighboring anchor positions (P3 and P9). Therefore, it has the same effect on HLA binding as previously reported spacing moieties like oligomers of 4-aminobutyrate or 6-aminohexanoate (20) or substituted phenanthridines for which a similar thermal destabilization (⌬Tm of ⫺12 °C) has been reported (21). However, a modification of TcR-binding amino acids that also enhances the binding affinity for the host MHC protein is possible. We describe here the first bifunctional spacer that provides additional interactions to the binding groove. The tetramer of HB, introduced between P3 and P9, significantly enhances binding to B*2705 (Table II). The beneficial effect of the (HB)4 spacer is attributed to two of its methyl substituents that reach the central pockets C/E of the binding cleft (Fig. 2). Since the global binding mode of the modified peptide has not been altered, the direct consequence of this replacement is an enhanced number of nonbonded contacts with the protein (Fig. 5). Again, discrepancies are observed for that series of compounds (ligands 7–12) between EC50 values and melting temperatures derived from CD experiments on the reconstituted complexes (Fig. 4). Tm values calculated for the tetrameric and trimeric HB analogues are nearly identical, whereas a 12–16-fold decreased binding was observed after shortening the length of the spacing area by one HB unit. The Tm values of a series of MHC-peptide complexes have recently been directly related to experimental equilibrium dissociation constants, KD (46). Thus, the higher affinity observed for the (HB)4 compounds relative to the parental peptide and the trimeric analogues could be due to faster on-rate kinetics. Alternatively, since the correlation proposed by Morgan et al. (46) takes into account a series of highly related nonapeptides, it may not be valid for altered ligands lacking a canonical nonapeptide structure. Importantly, the present study demonstrates that CD denaturing curves cannot be used alone to explain differences in binding of altered peptide ligands to a class I MHC protein. This is of crucial importance in any design effort aimed at enhancing binding affinities by increasing the on-rate kinetics of the designed molecule. It should be noted that two CD denaturation curves (peptides 7 and 12, Fig. 4) slightly deviate from the expected two-state model by presenting an additional transition at a temperature (45 °C) corresponding to the unfolding of peptide-free heavy chain (47). Such deviations from an ideal two-state model have already been observed (34) but remain difficult to explain at the molecular level. Our data demonstrate that B*2705-restricted epitopes may

be easily modified by introducing simple nonpeptidic elements in their central part without drastic changes in binding to their restriction MHC proteins. Two conditions seem to be necessary for these modifications: (i) the last amino acid (PC) should be a strong anchor, and (ii) the parent epitope should not contain a dominant anchor position between the P4 and P8 positions. Since this is the case for a majority of class I MHC peptide binding motifs (8), such chemical manipulations should be feasible for many antigenic peptides binding to class I MHC proteins. Class II MHC-binding peptides that utilize nearly all peptidic bonds to interact with their host MHC protein (48) must be excluded from these epitope modifications. The altered ligands reported in this study constitute a further step toward obtaining full nonpeptide ligands for class I MHC proteins. They represent interesting tools for altering the response of B*2705-restricted T cells to naturally occurring antigenic peptides and for designing novel synthetic vaccines. Acknowledgment—We thank the calculation center of the ETH Zu¨rich for allocation of computer time on the CRAY J90 and PARAGON supercomputers. REFERENCES 1. Heemels, M. T., and Ploegh, H. L. (1995) Annu. Rev. Biochem. 64, 643– 691 2. Madden, D. R. (1995) Annu. Rev. Immunol. 13, 587– 682 3. Madden, D. R., Gorga, J. C., Strominger, J. L., and Wiley, D. C. (1992) Cell 70, 1035–1048 4. Guo, H. C., Jardetzky, T. S., Garrett, T. P. J., Lane, W. S., Strominger, J. L., and Wiley, D. C. (1992) Nature 360, 364 –366 5. Saper, M. A., Bjorkman, P. J., and Wiley, D. C. (1991) J. Mol. Biol. 219, 277–319 6. Guo, H. C., Madden, D. R., Silver, M. L., Jardetzky, T. S., Gorga, J. C., Strominger, J. L., and Wiley, D. C. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8053– 8057 7. Falk, K., Ro¨tzschke, O., Stevanovic, S., Jung, G., and Rammensee, H.-G. (1991) Nature 351, 290 –296 8. Rammensee, H.-G., Friede, T., and Stevanovic, S. (1995) Immunogenetics 41, 178 –228 9. Jameson, S. C., Carbone, F. R., and Bevan, M. J. (1993) J. Exp. Med. 177, 1541–1550 10. Stryhn, A., Andersen, P. S., Pedersen, L. O., Svejgaard, A., Holm, A., Thorpe, C. J., Fugger, L., Buus, S., and Engberg, J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10338 –10342 11. Garcia, K. C., Degano, M., Stanfield, R. L., Brunmark, A., Jackson, M. R., Peterson, P. A., Teyton, L., and Wilson, I. A. (1996) Science 274, 209 –219 12. Garboczi, D. N., Ghosh, P., Utz, U., Fan, Q. R., Biddison, W. E., and Wiley, D. C. (1996) Nature 384, 134 –141 13. Brewerton, D. A., Hart, F. D., Nicholls, A., and Sturrock, R. D. (1973) Lancet 2, 994 –996 14. Benjamin, R., and Parham, P. (1990) Immunol. Today 11, 137–142 15. Hill, A. V. S., Elvin, J., Willis, A. C., Aidoo, M., Allsopp, C. E. M., Gotch, F. M., Gao, X. M., Takiguchi, M., Greenwood, B. M., Townsend, A. R. M., McMichael, A. J., and Whittle, H. C. (1992) Nature 360, 434 – 439 16. De Magistris, M. T., Alexander, J., Coggeshall, M., Altman, A., Gaeta, F. C. A., Grey, H. M., and Sette, A. (1992) Cell 68, 625– 634 17. Sloan-Lancaster, J., Evavold, B. D., and Allen, P. M. (1993) Nature 363, 156 –159 18. Ishioka, G. Y., Adorini, L., Guery, J.-C., Gaeta, F. C. A., LaFond, R., Alexander, J., Powell, M. F., Sette, A., and Grey, H. M. (1994) J. Immunol. 152, 4311– 4319 19. Kingsley, G., and Sieper, J. (1993) Immunol. Today 14, 387–391 20. Rognan, D., Scapozza, L., Folkers, G. and Daser, A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 753–757 21. Weiss, G. A., Collins, E. J., Garboczi, D. N., Wiley, D. C., and Schreiber, S. L. (1995) Chem. Biol. 2, 401– 407 22. Bouvier, M., and Wiley, D. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 4583– 4588 23. Pearlman, D. A., Case, D. A., Caldwell, J. C., Ross, W. S., Cheatham, T. E., Ferguson, D. M., Seibel, G. L., Singh, U. C., Weiner, P. K., and Kollman, P. A. (1995) AMBER 4.1, University of California, San Francisco 24. Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Jr., Ferguson, D. M., Spellmeyer, D. M., Fox, T., Caldwell, J. W., and Kollman, P. E. (1995) J. Am. Chem. Soc. 117, 5179 –5197 25. Fox, T., Scanlan, T. S., and Kollman, P. A. (1997) J. Am. Chem. Soc. 119, 11571–11577 26. Frisch, M. J., Trucks, G. W., Schlegel, H. B., Gill, P. M. W., Johnson, B. G., Robb, M. A., Cheeseman, J. R., Keith, T. A., Petersson, G. A., Montgomery, J. A., Raghavachari, K., Al-Laham, M. A., Zakrzewski, V. G., Ortiz, J. V., Foresman, J. B., Peng, C. Y., Ayala, P. A., Wong, M. W., Andres, J. L., Replogle, E. S., Gomperts, R., Martin, R. L., Fox, D. J., Binkley, J. S., Defrees, D. J., Baker, J., Stewart, J. P., Head-Gordon, M., Gonzalez, C., and Pople, J. A. (1995) Gaussian 94, Revision C.3, Gaussian, Inc., Pittsburgh, PA 27. Bayly, C. I., Cieplak, P., Cornell, W. D., and Kollman, P. A. (1993) J. Phys. Chem. 97, 10269 –10280 28. Cieplak, P., Cornell, W. D., Bayly, C., and Kollman, P. A. (1995) J. Comput.

Nonnatural HLA-B27 Ligands Chem. 16, 1357–1377 29. Rognan, D., Krebs, S., Kuonen, O., Lamas, J. R., Lo´pez de Castro, J. A., and Folkers, G. (1997) J. Comput. Aided Mol. Des. 11, 463– 478 30. Galocha, B., Lamas, J. R., Villadangos, J. A., Albar, J. P., and Lo´pez de Castro, J. A. (1996) Tissue Antigens 48, 509 –518 31. Ljunggren, H. G., Stam, N. J., Ohlen, C., Neefjes, J. J., Hoglund, P., Heemels, M. T., Bastin, J., Schumacher, T. N., Townsend, A., Karre, K., and Ploegh, H. L. (1990) Nature 346, 476 – 480 32. Ellis, S. A., Taylor, C., and McMichael, A. (1982) Hum. Immunol. 5, 49 –59 33. Nagai, K., and Thogersen, H. C. (1987) Methods Enzymol. 131, 266 –280 34. Weiss, G. A., Valentekovich, R. J., Collins, E. J., Garboczi, D. N., Lane, W. S., Schreiber, S. L., and Wiley, D. C. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 10945–10948 35. Bouvier, M., and Wiley, D. C. (1994) Science 265, 398 – 402 36. Privalov, P. L., and Gill, S. J. (1988) Adv. Protein Chem. 39, 191–234 37. Myers, J. K., Pace, C. N., and Scholtz, J. M. (1995) Protein Sci. 4, 2138 –2148 38. Rognan, D., Scapozza, L., Folkers, G., and Daser, A. (1994) Biochemistry 33, 11476 –11485 39. Jardetzky, T. S., Lane, W. S., Robinson, R. A., Madden, D. R., and Wiley, D. C. (1991) Nature 353, 326 –329 40. Ro¨tzschke, O., Falk, K., Stevanovic, S., Gnau, V., Jung, G., and Rammensee, H. G. (1994) Immunogenetics 39, 74 –77

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41. Reich, Z., Altman, J. D., Boniface, J. J., Lyons, D. S., Kozono, H., Ogg, G., Morgan, C., and Davis, M. M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 2495–2500 42. Garcia, F., Marina, A., and Lo´pez de Castro, J. A. (1996) Tissue Antigens 49, 215–221 43. Mu¨ller, H.-M., and Seebach, D. (1993) Angew. Chem. Int. Ed. Engl. 32, 477–502 44. Plattner, D. A., Brunner, A., Dobler, M., Mu¨ller, H.-M., Petter, W., Zbinden, P., and Seebach, D. (1993) Helv. Chim. Acta 76, 2004 –2033 45. Fields, G. B., and Noble, R. I. (1990) Int. J. Peptide Protein Res. 35, 161–214 46. Morgan, C. S., Holton, J. M., Olafson, B. D., Bjorkman, P. J., and Mayo, S. L. (1997) Protein Sci. 6, 1771–1773 47. Fahnestock, M. L., Johnson, J. L., Feldman, R. M. R., Tsomides, T. J., Mayer, J., Narhi, L. O., and Bjorkman, P. J. (1994) Biochemistry 33, 8149 – 8158 48. Stern, L. J., Brown, J. H., Jardetzsky, T. S., Gorga, J. C., Urban, R. G., Strominger, J. L., and Wiley, D. C. (1994) Nature 368, 215–221 49. Brooks, J. M., Murray, R. J., Thomas, W. A., Kurilla, M. G., and Rickinson, A. B. (1993) J. Exp. Med. 178, 897– 887 50. Huet, S., Nixon, D. F., Rothbard, J. B., Townsend, A., Ellis, S. A., and McMichael, A. J. (1990) Int. Immunol. 2, 311–316 51. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins Struct. Funct. Genet. 11, 281–296

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Articles Nonapeptide Analogues Containing (R)-3-Hydroxybutanoate and β-Homoalanine Oligomers: Synthesis and Binding Affinity to a Class I Major Histocompatibility Complex Protein Sorana Poenaru,‡,§ Jose´ R. Lamas,† Gerd Folkers,| Jose´ A. Lo´pez de Castro,† Dieter Seebach,*,‡ and Didier Rognan*,| Laboratory for Organic Chemistry, Swiss Federal Institute of Technology, Universita¨ tstrasse 16, CH-8092 Zu¨ rich, Switzerland, Centro de Biologia Molecular ”Severo Ochoa”, Facultad de Ciencias, Universidad Autonoma de Madrid, E-28049 Madrid, Spain, and Department of Pharmacy, Swiss Federal Institute of Technology, Winterthurerstrasse 190, CH-8057 Zu¨ rich, Switzerland Received December 4, 1998

Crystal structures of antigenic peptides bound to class I MHC proteins suggest that chemical modifications of the central part of the bound peptide should not alter binding affinity to the MHC restriction protein but could perturb the T-cell response to the parent epitope. In our effort in designing nonpeptidic high-affinity ligands for class I MHC proteins, oligomers of (R)-3-hydroxybutanoate and(or) β-homoalanine have been substituted for the central part of a HLA-B27-restricted T-cell epitope of viral origin. The affinity of six modified peptides to the B*2705 allele was determined by an in vitro stabilization assay. Four out of the six designed analogues presented an affinity similar to that of the parent peptide. Two compounds, sharing the same stereochemistry (R,R,S,S) at the four stereogenic centers of the nonpeptidic spacer, bound to B*2705 with a 5-6-fold decreased affinity. Although the chiral spacers do not strongly interact with the protein active site, there are configurations which are not accepted by the MHC binding groove, probably because of improper orientation of some lateral substituents in the bound state and different conformational behavior in the free state. However we demonstrate that β-amino acids can be incorporated in the sequence of viral T-cell epitopes without impairing MHC binding. The presented structure-activity relationships open the door to the rational design of peptide-based vaccines and of nonnatural T-cell receptor antagonists aimed at blocking peptide-specific T-cell responses in MHC-associated autoimmune diseases. Introduction Class I major histocompatibility complex (MHC)encoded proteins play a key role in the intracellular immune surveillance by selectively binding to intracellular peptide antigens and presenting them at the cell surface to T-cell receptors (TCRs) of cytotoxic T-lymphocytes (CTL).1 Due to the genetically encoded discrepancy between the limited number of class I alleles (about six) expressed by each individual and the infinite number of potential antigenic peptides (usually nonamers), class I MHC molecules must bind diverse sets of foreign peptides with a broad specificity but a high affinity. Numerous structural data on class I MHCpeptide complexes are nowadays available at the threedimensional level2 and provide an explanation for that paradigm. The 27 reported X-ray structures (for nine different class I MHC molecules) illustrate a peptide* To whom correspondence should be addressed. D. Seebach: fax, +41.1.632 11 44; e-mail, [email protected]. D. Rognan: fax, +41.1.635 68 84; e-mail, [email protected]. ‡ Laboratory for Organic Chemistry, SFIT. † Universidad Autonoma de Madrid. | Department of Pharmacy, SFIT. § Present address: Department of Chemistry, University of California, Berkeley, CA 94720-1460.

independent recognition in which both terminal ends of the peptide backbone are tightly bonded to conserved residues of the MHC binding groove. Allele specificity is ensured by the interaction of anchoring side chains,3,4 usually at positions P2, P3 (Pn standing for position n), and the C-terminus with polymorphic pockets5 of the host MHC protein. The central part of the bound peptides (from positions 4 to 8) generally zigzags6 or bulges7 out of the binding groove and thus allows variation in the length of the bound peptides (from 8 to 11 amino acids). Systematic peptide mutation8 and X-ray structure of MHC-peptide-TCR ternary complexes9-12 show that this central part whose conformation is not complementary to that of the MHC protein is the major contact area for Rβ TCRs that trigger the T-cell response to the foreign peptide. The tight association observed between MHC expression and susceptibility or resistance to autoimmune disorders led us to consider class I MHC proteins as particularly interesting targets for the selective immunotherapy of autoimmune diseases. At least two ways of shunting the T-cell response to autoantigens using small-molecular-weight molecules have been proposed. The first one involving MHC blockade by a high-affinity

10.1021/jm981123l CCC: $18.00 © 1999 American Chemical Society Published on Web 06/08/1999

Nonapeptide Analogue Binding Affinity to MHC Protein

Journal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2319

competitor13 is unlikely as MHC-bound peptides at the cell surface are almost impossible to displace.14 The only way to overcome this drawback would be to supply the peptide competitor in liposomes15 or as lipopeptides16 into the endoplasmic reticulum where assembly of the class I MHC-peptide complexes takes place. The second inhibiting pathway relying on TCR antagonism17 suggests that the presentation of the epitope to autoreactive T-cells would be antagonized by a modified peptide analogue. This approach is much more promising since only a few TCRs at the surface of CTLs need to be targeted,18 whereas MHC blockade requires saturation of all MHC binding sites at the cell surface.19 Two prerequisites are however necessary for designing TCR antagonists: (i) a good affinity to the MHC-restriction protein, (ii) a fast dissociation of the corresponding MHC-ligand complex to the TCR.20,21 The few TCR antagonists known to date are all peptide analogues for which one TCR-anchoring amino acid has been mutated.22 Unfortunately, the poor stability and pharmacokinetic properties inherent to their peptidic nature preclude their general use as immunosuppressors. Thus, there is a need for designing high-affinity nonpeptide ligands for class I MHC proteins. Rather few variations around the canonical nonapeptide structure have been described up to date.23 Peptides bearing unnatural Lor D-R-amino acids at MHC-anchoring positions,24-29 reduced peptide bond pseudopeptides,30 retroinverso analogues,31 poly-N-acylated amines,32 or incorporation of a β-homoglycine residue at the peptide N-terminus33 have been reported. An alternative strategy we initiated 3 years ago is to replace the central TCR-binding amino acids by various nonpeptidic spacers: oligomers of aminoalkanoates,24,34 phenanthridine derivatives,35 or poly(ethylene glycol) loops.36 All these chemical modifications led to ligands that could associate with class I MHC proteins but always with a slight decrease in binding affinity when compared to that of the parent peptides. We recently described the replacement of a natural pentameric peptide sequence (from positions P4 to P8) by (R)-3-hydroxybutanoate (R-HB) oligomers in HLAB27-binding nonapeptides37 while enhancing 5-fold the binding affinity for the MHC restriction protein.34 However, the partial hydrolysis of oligo-HB ester bonds, observed during the synthesis, suggests that these analogues should have very poor in vivo pharmacokinetic properties because of their high sensitivity to esterases and peptidases. Recent reports on the remarkable enzymatic stability of β-peptides38,39 led us to consider oligomers of β-amino acids as potential surrogates for the TCR-binding residues of class I MHCbinding peptides. Since β-homoalanine (β-HAla) is an isostere of HB, binding to HLA-B27 should thus be retained in light of our previous results on poly(ester peptides) (PEPs).34 However, the low solubility of protected β-HAla oligomers in any solvent40 could be a drawback to the synthesis and biological evaluation of these compounds. To increase the solubility in water of compounds containing four β-HAla units,41 a positively charged peptide epitope from the HIV-1 gp120 protein (G314RAFVTIGK322, one-letter amino acid code), known to bind well to HLA-B*2705,34 was chosen as template for the reported chemical modifications (Table 1). Fur-

Scheme 1a

a (a) DCC, DMAP, CH Cl ; (b) HCl/ether (satd); (c) H , Pd/C, 2 2 2 MeOH; (d) Boc-Ala-OH, HOBt, EDC, Et3N, CH2Cl2; (e) H2, Pd/C, MeOH; (f) HCl•H-Lys(Z)-OBn, HOBt, EDC, Et3N, CH2Cl2; (g) HCl/ dioxane (satd); (h) HOBt, EDC, DIEA, CH2Cl2; (i) TFA/CH2Cl2, 1:1.

thermore, combining β-HAla and HB oligomers should enhance the solubility of the resulting spacers in chlorinated solvents42 and thus facilitate their synthetic access. Most of the designed peptide analogues bind indeed with a high affinity to a class I MHC protein (HLA-B*2705 allele), whose expression is associated with susceptibility to severe autoimmune diseases.43 Results and Discussion Chemistry. Synthesis of the derivative 11 was achieved using a fragment-type coupling strategy (Scheme 1). Boc-protected β-homoalanine38,44 and benzyl 3-hydroxybutanoate (2)45 were coupled using DCC and DMAP as activating reagents46 to give 3. Deprotection of the amino group under acidic conditions gave the amino ester 4, whereas hydrogenolysis of the benzyl ester gave acid 5. Coupling of 4 with the commercially available Boc-protected alanine under traditional HOBt/ EDC peptide conditions47-49 gave the fully protected derivative 6 whose benzyl ester group was cleaved by H2 (Pd/C) to yield acid 7. 1H NMR measurements led to assignment of all signals to the corresponding protons of the R-amino acid, of the HB unit, and of the β-HAla moiety. To obtain the second fragment 9 with free amino and protected carboxy group, the acid 5 was coupled with H-Lys(Z)-OBn, using the HOBt/EDC strategy to give 8 (80%), treatment of which with a saturated HCl/dioxane solution yielded the corresponding HCl salt 9. Compound 10, consisting of six building blocks, was then

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Scheme 2a

Poenaru et al.

Scheme 4a

a (a) HCl•H-Lys(Z)-OBn, HOBt, EDC, Et N; (b) HCl/dioxane 3 (satd); (c) 1, HOBt, EDC, DIEA, DMF; (d) HCl/dioxane (satd); (e) Fmoc-Ala-OH, DCC, DMAP, CH2Cl2; (f) TFA/CH2Cl2, 1:1; (g) HOBt, EDC, DIEA, CH2Cl2/DMF, 1:1; (h) Et2NH/DMF, 1:4.

Scheme 3a

a (a) Boc-Arg(NO )-OH, HOBt, EDC, DIEA, DMF; (b) TFA, 10 2 min; (c) Boc-Gly-OH, HOBt, EDC, DIEA, DMF; (d) H2, Pd/BaSO4, TFE/AcOH, 4:1; (e) TFA, 10 min.

a (a) HCl•H-Lys(Z)-OBn, HOBt, EDC, DIEA, DMF; (b) TFA, 10 min; (c) Boc-Ala-OH, HOBt, EDC, DIEA, DMF; (d) TFA, 10 min.

obtained in 90% yield by coupling of 7 with 9.50 Subsequent cleavage of the Boc protecting group led to the amino ester 11 which was used for the next coupling with arginine, without further purification (Scheme 4). The derivative 20, with HB and β-HAla incorporated in different sequence (Scheme 2), was synthesized by

application of the same fragment coupling strategy. The fragment 15 was synthesized in a linear fashion, coupling first Boc-β-HAla-OH (1) with H-Lys(Z)-OBn to give dipeptide 12 (85%) which was deprotected to give the HCl salt 13 (HCl in dioxane). The amino functionality of 13, set free in situ by the base present in the reaction mixture, was coupled with 1 to give the fully protected compound 14 (74% from 12). The Boc group of 14 was cleaved to yield 15. The fragment 17 was obtained by ester formation between the hydroxy dimer 1637,51 and Fmoc-protected alanine (using DCC, DMAP), and cleavage of the tert-butyl ester group (50% TFA) gave the desired compound 18. It should be noted that by using a small amount of DMAP (0.05 equiv), no racemization was observed upon coupling.37 The amino functionality, generated by in situ deprotonation of the HCl salt 15, was coupled with the acid group of 18 (HOBt/EDC) to give the fully protected compound 19 in 92% yield. The Fmoc protecting group was then cleaved (20% diethylamine in DMF52) to give the amino ester 20. It is noteworthy that the backbone of compound 19 varies from that of 10 only by the respective positions of HB and β-HAla in the sequence. However their respective solubilities in organic solvents are very different. Compound 10 is highly soluble in chlorinated

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Table 1. Binding and Analytical Properties of Ligands 27, 28, and 29a-d compd

sequence

C50a (µmol)

HPLC (tR, min)b

MSc (M + 1)+

HIV gp120d A68P1e 27 28 29a 29b 29c 29d

Gly-Arg-Ala-[Phe-Val-Thr-Ile-Gly]-Lys Glu-Val-Ala-Pro-Pro-Glu-Tyr-His-Arg Gly-Arg-Ala-[(S-β-HAla-R-HB)2]-Lysg Gly-Arg-Ala-[(R-HB)2-(S-β-HAla)2]-Lys Gly-Arg-Ala-[(R-β-HAla)4]-Lys Gly-Arg-Ala-[(S-β-HAla-R-β-HAla)2]-Lys Gly-Arg-Ala-[(S-β-HAla)4]-Lys Gly-Arg-Ala-[(R-β-HAla)2-(S-β-HAla)2]-Lys

2.8 nbf 2.8 17.0 2.8 4.8 6.0 30.0

18.7 19.5 16.4 15.4 15.3 15.4

773.3 774.1 771.9 772.6 771.9 772.0

a Concentration of ligand at which HLA-B*2705 fluorescence (measured by FMC analysis with an anti-B27 monoclonal antibody) on RMA-S cells was half the maximum obtained with that compound (see Experimental Section). b HPLC purification using a gradient of A (0.1% trifluoroacetic acid in water) and B (acetonitrile): 0-100% B, 60 min. c MALDI-TOF spectra, recorded on a Bruker Biflex instrument (Bruker-Franzen Analytik, Bremen, Germany) in linear mode. d HIV-1 glycoprotein 120 (314-322). e Self-peptide eluted from the HLAA68 allotype.8 f No detectable binding at 10-4 M. g β-HAla, β-homoalanine; HB, 3-hydroxybutanoate.

solvents such as CH2Cl2, whereas 19 is poorly soluble even in DMF. The configurational isomers (diastereoisomers) 21a-d containing four β-HAla units (Scheme 3) have been previously prepared, starting from (S)- and (R)-Boc-AlaOH and using a fragment coupling strategy.40 These oligomers are difficult to handle because of their low solubility in any solvent tested so far. For example, the 1H NMR spectra of protected β-HAla tetramers can only be measured using dimethyl-d6 sulfoxide as solvent and show rather broad signals. During the synthesis, larger amounts of DMF were necessary to dissolve the β-HAla oligomers (c ) 0.05 M), as compared to those required in R-peptide synthesis, and reaction times were consequently longer. Therefore, the yields of the reactions are not specified, since no purification is possible before the last deprotection step, due to the poor solubility of this class of compounds. The acids 21 were coupled with H-Lys(Z)-OBn using HOBt/EDC, to provide the fully protected derivatives, the Boc protecting groups of which were cleaved (concentrated TFA) to give the TFA salts 22 which were precipitated with ether and dried in high vacuum before the next reaction step: deprotonation and reaction with Boc-Ala-OH to yield, after another deprotection step, the corresponding TFA salts 23. During the cleavage of the Boc group, the Z protecting group of the lysine side chain was partially cleaved, giving rise to a byproduct (e5%) which has been detected by mass spectrometry. Such debenzylations have been previously reported by Merrifield et al.53 However, considering the much harsher conditions used by the Merrifield group, the observed loss of the Z group, in our case, was surprising. Due to solubility problems, it was impossible to purify the intermediates at this stage. Thus, we have carried the byproduct all along the following synthetic steps, with the consequence that some additional impurities were formed. After the last deprotection step, preparative HPLC purification still gave the desired pure compounds (27-29). The amino functionalities of the derivatives 11, 20, and 23a-d (Scheme 4) were allowed to react with Boc-Arg(NO2)OH,54 using the same coupling procedure as for the other coupling steps. Again, the Boc groups of the fully protected derivatives were cleaved with TFA to yield compounds 24, 25, and 26a-d which, in turn, were coupled with Boc-Gly-OH to give the fully protected nonapeptides analogues. The NO2 and Z protecting groups, as well as the benzyl ester group, were then simultaneously removed by hydrogenation, using Pd/

Figure 1. Dynamic properties of complexes of B*2705 with two modified peptides (29b, 29d) and the reference HIV-1 gp120 (314-322) peptide. (A) Intermolecular hydrogen-bond frequencies, recorded for the whole 500-ps trajectory of HLAB27-ligand complexes over 1000 conformations. Frequencies between 25% and 50% and higher than 50% are displayed as white and gray columns, respectively. (B) Buried surface areas of HLA-B27-bound ligands 29b (white columns) and 29d (gray columns) calculated on energy-minimized time-averaged conformations. Surface areas were calculated using the MS program74 with a 1.4-Å radius probe.

BaSO4 as catalyst.56 Subsequent treatment with TFA led to cleavage of the Boc groups to give, after HPLC purification, the desired nonapeptide analogues 27, 28, and 29a-d which were used for binding assays. Binding Affinity to HLA-B*2705. The binding affinities of the modified peptides clearly show that the chirality of the spacer is important for recognition of the B*2705 protein. Compounds in which the chiral building block linked with the C-terminus (PC) has (R)configuration (27, 29a-b) were all potent ligands with

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binding affinities similar to that of the parent HIV-1 gp120 peptide (Table 1). This observation is in agreement with our previous report on PEPs for which a penultimate (R)-HB unit was proposed to interact with the MHC binding groove.34 Analogues bearing a moiety of (S)-configuration next to PC were less active, especially compounds 28 and 29d sharing the same sequence of (R,R,S,S)-configuration of the spacing oligomers (Table 1). However, an (S)-chiral spacer attached to PC does not necessarily prevent binding (see compound 29c, Table 1). Furthermore, it seems that certain configurations of the four spacing monomers are detrimental to binding. Thus, the two weakest binders (28, 29d) share the same (R,R,S,S)-configuration at positions 4 to 7. Replacing ester by amide groups in the spacer (cf. 27 with 29b and 28 with 29d) did not affect binding for both high-affinity and low-affinity ligands. This result is in agreement with the available X-ray structure56 of a B*2705-nonapeptide complex, showing weak contributions of peptide bonds, located between the P4 and P8 positions, to the binding of a nonapeptide to HLAB27. Apart from binding potencies, it should be noticed that HB-containing compounds 27 and 28 are probably still sensitive to esterases, although stability studies on these compounds have not been performed yet. We also expect that the replacement of HB oligomers by β-amino acids in analogues 29a-d enhances the resistance of the modified peptides to enzymatic degradation. Molecular Modeling of B*2705-Ligand Complexes. To find a rational explanation for the weak binding of compounds with (R,R,S,S)-configuration of the chiral spacer, a 500-ps molecular dynamics (MD) study of the binary complexes between B*2705 and three ligands was undertaken. Compound 29b was chosen as representative of the high-affinity peptides, whereas 29d was selected as representative of weak binding ligands. The parent HIV peptide was selected as reference. The trajectory of the three solvated complexes was stable after 350 ps, with rms deviations of the protein backbone from the starting X-ray coordinates of ca. 1.5 Å (data not shown). We previously used atomic fluctuations of the bound ligands, as a criterion, for discriminating high-affinity from low-affinity peptides.24,33,57 In the present case, they were very similar for ligands 29b and 29d. Thus, subtle differences must cause the very different binding affinities of the two modified peptides. In fact, recording the frequency of the MHC-ligand hydrogen bonds allows to distinguish the two modified peptides. High-affinity ligands (HIV gp120, 29b) present many more hydrogen bonds to the HLA-B27 binding groove than the weak binding compound 29d (Figure 1A). Strong H-bonds with a frequency higher than 50% were remarkably identical in both cases, but medium H-bonds (with frequencies between 25% and 50%) are significantly in favor of 29b. A very similar pattern has already been observed for a set of four natural peptides binding to two closely related HLA-B27 alleles.33 The major differences be-

tween the two nonnatural complexes could be correlated with the H-bond-donating strength of the N-terminus, well-known to significantly contribute to the binding free energy of nonapeptides to class I MHC proteins.58 The buried surface areas of each monomer of the protein-bound ligand were also very similar with the exception of two residues P5, and PC (Figure 1B). P5 corresponds to the second unit of the spacer (R-βHAla in both cases). Depending on its environment in the sequence of the modified peptide, it is more or less deeply buried in the HLA-B27 binding groove. With compound 29b, the P5 position is significantly deeper inside the groove than with compound 29d (compare Figure 2A,B). However, this feature is unlikely to induce nearly a 10-fold difference in the binding affinity of the corresponding ligands. The C-terminal amino acid (Lys) also shows a better fit in the case of the high-affinity ligand (Figure 1B). As the C-terminal residue is an important anchor to B*2705,56 this structural difference should also contribute to the improved binding of 29b versus 29d. However, the computed properties of the two analogues bound to their target protein can only explain a part of the experimentally determined difference of binding affinities. The modeling study presented here takes into account only enthalpic contributions to the binding of each ligand to HLA-B*2705. As desolvation energies and rotational/translational entropy losses upon binding (assuming a conserved binding mode of the two compounds) should be very similar due to the structural analogy of all modified peptides listed in Table 1, the 10-fold decreased binding of two analogues (28, 29d), having the same configuration, may be due to different association rates and different conformational populations in the free state. This feature has already been experimentally described for two related PEPs,34 for which the length of the polyester spacer varies. Hydrophobic β-peptides are known to have conformations strongly depending on the chirality of their residues and on the nature of their side chains.38,59 The (R,R,S,S)-configuration of four chiral monomers in low-affinity ligands might lead to a conformational space arrangement in the free state that is different from that of high-affinity compounds (27, 28, 29a-c). The higher “strain energy” necessary to bring ligands 28 and 29d from the free to the bound state may partially contribute to the weaker binding of these two analogues. Unfortunately, simulating the free ligands, although computationally easier, is very risky because they adopt no stable secondary structure, as concluded from their CD or NMR spectra. Conclusion Replacing the central amino acids of class I MHCbinding peptides by (R)-3-hydroxybutanoate and(or) β-homoalanine oligomers leads to still high-affinity ligands. Up to now, β-amino acids have hardly been used in medicinal chemistry. Some natural β-amino acids (taurine, β-aminobutyric acid, β-aminoisobutyric acid)

Figure 2. Three-dimensional structure of HLA-B*2705 in complex with 29b (A) and 29d (B). Peptide positions are labeled at the CR atom from 1 (P1) to 8 (P8). The backbone trace of the MHC antigen-binding domain (R1, R2) of the B*2705 protein is represented as bands (R helices), arrows (β strands), and tubes (coil). Altered peptides are displayed by a ball-and-stick model. A white arrow indicates the position of the second β-ΗAla unit in both chiral spacers. The figure has been prepared using the MOLSCRIPT75 and RASTER3D76 programs.

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have been reported as agonists of the inhibitory glycine receptor.60 Substituted β-amino acids have also been described as fibrinogen receptor GIIb/IIIa antagonists,61 β-lactamase inhibitors,62 µ-opioid receptor agonists,63 or enkephalin-degrading enzyme inhibitors.64 Furthermore, various β-amino acids are found in natural antibiotics, fungicides, and antineoplastic compounds.65 However, to the best of our knowledge, this is the very first report of biologically active molecules containing β-amino acid oligomers. The present study demonstrates that β-amino acids are valuable tools, indeed, for designing peptidomimetics of bioactive peptides. By contrast to most R-amino acid surrogates, the H-bonding properties of backbone atoms, the backbone direction, or the side chain directionality might be similar in natural and β-peptides, at the condition that the β-peptide can adopt the biologically active conformation of its natural R-peptide analogue. Thus, if all side chains are not mandatory for biological activity, β-amino acids and, more generally, β-peptides undoubtedly represent new promising tools in medicinal chemistry. In the special case of class I MHC ligands, one might imagine to use β-amino acids for replacing MHC anchors and(or) TCR contact residues. Such altered peptides may thus lead to peptide-based vaccines and TCR antagonists, which would be stable to all common peptidases tested so far, including pronase, 20S proteasome, and proteinase K.

DIEA, 3 equiv). HOBt (1.25 equiv), the acid (1 equiv), and EDC (1.25 equiv) were then successively added. The reaction mixture was allowed to warm to room temperature and then stirred for 18 h. The mixture was diluted with CH2Cl2 and washed with 1 N HCl, saturated NaHCO3 solution, and brine. The organic layer was dried over anhydrous MgSO4, filtrated, and concentrated. The resulting residue was purified on silica gel to afford the pure product. General Procedure B: Amino Acid Coupling. The free amine or the appropriate salt (1 equiv) was dissolved in DMF (0.15 M) under argon and cooled to 0 °C. The reaction mixture was treated with DIEA (3 equiv). HOBt (1.25 equiv), the acid (1 equiv), and EDC (1.25 equiv) were then successively added. The product was precipitated by the addition of a saturated NaHCO3 solution. The precipitate was washed several times with saturated NaHCO3, 1 M KHSO4 solutions and H2O and dried 24 h under high vacuum to give the crude product which was utilized in the next step without further purification. General Procedure C: Boc Cleavage Using a HCl Solution. Under argon and at 0 °C, the Boc-protected compound was dissolved in a saturated HCl/(EtO2 or dioxane) solution. The mixture was stirred for 15 min to 1 h and then evaporated. The resulting HCl salt was precipitated in ether, dried under high vacuum, and used for the next step without further purification. General Procedure D: Boc Cleavage Using a TFA Solution. Under argon and at 0 °C, the Boc-protected compound was dissolved in a TFA/CH2Cl2 (50-100%) solution. The mixture was stirred for 10 min to 1 h and then evaporated. The resulting TFA salt was precipitated in Et2O, dried under high vacuum, and used for the next step without further purification. General Procedure E: Final Deprotection. The fully protected compound was dissolved in TFE/CH3COOH (3/1), and a catalytic amount of 10% Pd/BaSO4 was added. The apparatus was evacuated and flushed three times with H2, and the mixture was stirred under an atmosphere of H2 for ca. 15 h. The mixture was then filtrated though Celite, concentrated, and precipitated from Et2O. The resulting yellow-white CH3COOH salt was then treated with concentrated TFA. After 15 min, the crude product was precipitated from Et2O and purified by HPLC. Boc-S-β-HAla-R-HB-OBn (3). To a solution of the (R)-3hydroxybutanoic benzyl ester (2)45 (1.90 g, 9.8 mmol) in CH2Cl2 (30 mL) was added a solution of the acid 138 (2.00 g, 9.8 mmol) in CH2Cl2 (30 mL) under argon, cooled to -5 °C. DCC (2.12 g, 10.3 mmol) and DMAP (0.09 g, 0.49 mmol) were added. The resulting mixture was allowed to warm to room temperature and then stirred for 24 h. The mixture was diluted with Et2O and washed with 1 N HCl, saturated NaHCO3 solution, and brine. The organic layer was dried over anhydrous MgSO4, filtrated, and concentrated. The residue was purified on silica gel (20% Et2O/pentane) and gave compound 3 (3.25 g, 88%) as a colorless oil. 1H NMR (300 MHz, CDCl3): δ 7.37-7.33 (m, 5H ar), 5.36-5.26 (m, 1H, CHO), 5.14 (AB, J ) 12.1, 1H, OCH2Ph), 5.09 (AB, J ) 12.1, 1H, OCH2Ph), 5.00-4.90 (m, 1H, NH), 4.10-3.96 (m, 1H, CHN), 2.66 (dd, ABX, J ) 7.5, 15.6, 1H, CH2CHO), 2.54 (dd, ABX, J ) 5.3, 15.6, 1H, CH2CHO), 2.43 (dd, ABX, J ) 5.3, 14.9, 1H, CH2CHN), 2.37 (dd, ABX, J ) 5.9, 14.9, 1H, CH2CHN), 1.43 (s, 9H, tBu), 1.29 (d, J ) 6.2, 3H, Me of HB), 1.17 (d, J ) 6.5, 3H, Me of β-HAla). 13C NMR (75 MHz, CDCl ): δ 171.00, 170.42, 155.36, 135.93, 3 128.84, 128.60, 67.58, 66.65, 43.60, 40.77, 28.46, 20.31, 19.96. FAB-MS: m/z 759 {26, (2M + 1)+}, 308 {64, (M + 1)+}, 280 (100). HCl•H-S-β-HAla-R-HB-OBn (4). According to general procedure C, compound 3 (227 mg, 0.6 mmol) was treated with a saturated HCl/Et2O solution (6 mL). The resulting HCl salt 4 was obtained as a white precipitate and used in the next coupling step without further purification. Boc-S-β-HAla-R-HB-OH (5). The benzyl-protected compound 3 (900 mg, 2.9 mmol) was dissolved in MeOH (20 mL); catalytic amounts of 10% Pd/C (90 mg) and acetic acid (0.1

Experimental Section Abbreviations: (R)-β-homoalanine (R-β-HAla), (S)-β-homoalanine (S-β-HAla), dicyclohexylcarbodiimide (DCC), diisopropylethylamine (DIEA), 4-(dimethylamino)pyridine (DMAP), dimethylformamide (DMF), N-(3-(dimethylamino)propyl)-N′ethylcarbodiimide hydrochloride (EDC), 1-hydroxy-1H-benzotriazole (HOBt), trifluoroacetic acid (TFA), trifluoroethanol (TFE), (R)-3-hydroxybutanoate (R-HB). Chemistry. Dichloromethane (CH2Cl2) was dried over 4-Å molecular sieves. Solvents for chromatography and workup procedures were distilled from Sikkon (anhydrous CaSO4, Fluka). Triethylamine (Et3N) was distilled from CaH2 and stored over KOH. Amino acid derivatives were purchased from Bachem or Senn. All other reagents were used as received from Fluka. 1H (300-MHz) and 13C (75-MHz) NMR spectra were recorded on a Varian Gemini 300 spectrometer and are reported in ppm on the δ scale from TMS. Coupling constants are reported in hertz (Hz). FAB-MS spectra were obtained with a Hitachi Perkin-Elmer RHU-6M using a 3-nitrobenzyl alcohol (3NOBA) matrix. Elemental analyses were performed by the Microanalytical Laboratory of the Laboratorium fu¨r Organische Chemie, ETH-Zu¨rich (only analyses above 0.4% were given). Chromatography generally refers to flash silica gel 60 (Fluka 40-63 mm) and TLC (Merck Kieselgel 60 F254 plates), detection with UV and ninhydrin. HPLC analyses were carried out on a C18 analytical column on a Knauer HPLC system (pump type 64, EuroChrom 2000 integration package, degaser, UV detector (variable-wavelength monitor)) using a linear gradient of (A) 0.1% CF3COOH in H2O and (B) MeCN at a flow rate of 1 mL/min with UV detection at 220 nm. HPLC purification was carried out on a C8 preparative column on a Knauer HPLC system (pump type 64, programmer 50, UV detector (variable-wavelength monitor)) using a gradient of (A) 0.1% CF3COOH in H2O and (B) MeCN at a flow rate of 4 mL/min with UV detection at 214 nm. Retention times (tR) are given in min. General Procedure A: Amino Acid Coupling. The free amine or the appropriate salt (1 equiv) was dissolved in CH2Cl2 or 50% CH2Cl2/DMF (0.1 M) under argon and cooled to 0 °C. The reaction mixture was treated with a base (Et3N or

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mL) were added. The apparatus was evacuated and flushed three times with H2, and the mixture was stirred under an atmosphere of H2 for ca. 8 h. Subsequent filtration though Celite and concentration under reduced pressure yielded the acid 5 (558 mg, 84%) as a colorless oil which was identified by NMR and used for the next coupling step without purification. Boc-Ala-S-β-HAla-R-HB-OBn (6). According to general procedure A, to a solution in CH2Cl2 (26 mL) of HCl salt 4 (1 equiv, 2.61 mmol) was added Et3N (1.45 mL, 10.4 mmol). HOBt (440 mg, 3.3 mmol), Boc-Ala-OH (4.94 mg, 2.61 mmol), and then EDC (623 mg, 3.3 mmol) were successively added to the reaction. The residue was purified by recrystallization (Et2O/ pentane, 2/5) to give compound 6 (994 mg, 85%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 7.35-7.326 (m, 5H ar), 6.70 (br d, J ) 6.8, 1H, NH), 5.33-5.27 (m, 1H, CHO), 5.12 9 (s, 2H, OCH2Ph), 5.12-5.06 (m, 1H, NH), 4.36-4.22 (m, 1H, CHN), 4.18-4.06 (m, 1H, CHN), 2.74-2.66 (m, 1H, CH2CHO), 2.58 (dd, ABX, J ) 5.0, 15.57, 1H, CH2CHO), 2.42 (d, J ) 5.3, 2H, CH2CHN), 1.44 (s, 9H, tBu), 1.34-1.29 (m, 6H, 2 Me), 1.17 (d, J ) 6.8, 3H, Me). 13C NMR (75 MHz, CDCl3): δ 172.12, 170.87, 135.80, 128.86, 128.67, 128.62, 67.87, 66.78, 42.02, 40.69, 40.33, 28.38, 19.98, 19.66, 18.64. FAB-MS: m/z 901 {11, (2M + 1)+}, 451 {100, (M + 1)+9}, 351 (48). Boc-Ala-S-β-HAla-R-HB-OH (7). The benzyl-protected compound 6 (675 mg, 1.5 mmol) was dissolved in MeOH (10 mL); catalytic amounts of 10% Pd/C (70 mg) and acetic acid (0.1 mL) were added. The apparatus was evacuated and flushed three times with H2, and the mixture was stirred under an atmosphere of H2 for ca. 8 h. Subsequent filtration though Celite and concentration under reduced pressure yielded the acid 7 in almost quantitative yield as a colorless oil which was used for the next coupling step without purification. Boc-S-β-HAla-R-HB-Lys(Z)-OBn (8). According to general procedure A, to a solution in CH2Cl2 (15 mL) of HCl‚H-Lys(Z)-OBn (784 mg, 1.9 mmol) was added Et3N (1.08 mL, 7.7 mmol). HOBt (326 mg, 2.4 mmol), compound 5 (558 mg, 1.9 mmol), and then EDC (460 mg, 2.4 mmol) were successively added to the reaction. The residue was purified on silica gel (50% Et2O/pentane) to give compound 8 (964 mg, 78%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 7.38-7.29 (m, 10H ar), 6.96-6.90 (m, 1H, NH), 5.27-5.17 (m, 1H, CHO), 5.225.09 (m, 2H, OCH2Ph), 5.08 (s, 2H, OCH2Ph), 4.97-4.94 (m, 1H, NH), 4.90-4.86 (m, 1H, NH), 4.66-4.58 (m, 1H, CHN), 4.16-4.05 (m, 1H, CHN), 3.16-3.09 (m, 2H, CH2NHZ), 2.542.27 (m, 4H, CH2CHN, CH2CHO), 1.90-1.60 (m, 4H, 2 CH2), 1.50-1.28 (m, 2H, CH2), 1.40 (s, 9H, tBu), 1.30 (d, J ) 6.2, 3H, Me), 1.13 (d, J ) 6.8, 3H, Me). 13C NMR (75 MHz, CDCl3): δ 172.57, 170.84, 169.74, 156.83,155.65, 136.87, 135.69, 128.86, 128.76, 128.71, 128.55, 111.19, 79.61, 68.19, 67.16, 66.74, 52.19, 43.84, 42.30, 41.96, 40.61, 31.71, 29.46, 28.43, 22.42, 20.90, 19.46. FAB-MS: m/z 642 {12, (M + 1)+}, 542 (100). HCl•H-S-β-HAla-R-HB-Lys(Z)-OBn (9). According to general procedure C, compound 8 (712 mg, 1.1 mmol) was treated with a saturated HCl/dioxane solution (10 mL). After 15 min, the reaction was completed and the solvent was evaporated. The resulting HCl salt 9 was obtained in almost quantitative yield as a white precipitate and used in the next coupling step without further purification. Boc-Ala-(S-β-HAla-R-HB)2-Lys(Z)-OBn (10). According to general procedure A, to a solution in CH2Cl2 (15 mL) of the HCl salt 9 (1 equiv, 1.5 mmol) was added DIEA (1.0 mL, 6.0 mmol). HOBt (253 mg, 1.8 mmol), compound 7 (1 equiv, 1.5 mmol), and then EDC (358 mg, 1.8 mmol) were successively added to the reaction. The residue was purified on silica gel (ethyl acetate/hexane, 9/1) to give compound 10 (884 mg, 90%) as a white solid foam. 1H NMR (300 MHz, CDCl3): δ 7.367.30 (m, 10H ar), 7.10-7.00 (m, 3H, NH), 5.34 (d, J ) 7.5, 1H, NH), 5.30-5.10 (m, 3H, CHO, NH), 5.21-5.09 (m, 2H, OCH2Ph), 5.07 (s, 2H, OCH2Ph), 4.60-4.53 (m, 1H, CHN), 4.444.31 (m, 2H, CHN), 4.17-4.08 (m, 1H, CHN), 3.16-3.08 (m, 2H, CH2NHZ), 2.55-2.29 (m, 8H, CH2CHN, CH2CHO), 1.881.60 (m, 2H, CH2), 1.58-1.22 (m, 4H, CH2), 1.42 (s, 9H, tBu), 1.31 (d, J ) 6.8, 3H, Me), 1.29 (d, J ) 6.2, 3H, Me), 1.25 (d, J

) 6.2, 3H, Me), 1.17 (d, J ) 6.8, 3H, Me), 1.15 (d, J ) 6.5, 3H, Me). 13C NMR (75 MHz, CDCl3): δ 172.49, 170.79, 170.08, 169.58, 156.98, 136.84, 135.64, 128.86, 128.78, 128.73, 128.54, 128.37, 128.29, 68.61, 68.27, 67.19, 66.74, 52.39, 42.47, 42.21, 41.24, 40.40, 31.42, 29.40, 28.41, 22.36, 20.06, 19.85, 19.71, 18.81. FAB-MS: m/z 906 {6, (M + Na)+}, 884 {32, (M + 1)+}, 784 (100). Anal. (C45H65N5O13•H2O) C, H, N. TFA•H-Ala-(S-β-HAla-R-HB)2-Lys(Z)-OBn (11). According to general procedure D, compound 10 (654 mg, 0.74 mmol) was treated with a CH2Cl2/TFA (1:1) solution (6 mL). After 30 min, the reaction was completed and the solvent was evaporated. The TFA resulting salt 11 was obtained in almost quantitative yield as a white precipitate (from Et2O) and used in the next coupling step without further purification. Boc-S-β-HAla-Lys(Z)-OBn (12). According to general procedure A, to a solution in DMF (50 mL) of the HCl salt of Lys(Z)-OBn (2.00 g, 4.9 mmol) was added Et3N (2.04 mL, 14.7 mmol). HOBt (0.83 g, 6.1 mmol), the acid Boc-β-HAla-OH (1) (1.00 g, 4.9 mmol), and then EDC (1.17 g, 6.1 mmol) were successively added to the reaction. The residue was purified by recrystallization (ethyl acetate/hexane, 20/1) to give compound 12 (2.30 g, 85%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 7.37-7.26 (m, 10H ar), 6.48-6.36 (m, 1H, NH), 5.22-5.10 (m, 3H, OCH2Ph, NH), 5.09 (s, 2H, OCH2Ph), 4.954.87 (m, 1H, NH), 4.62-4.56 (m, 1H, CHN), 4.00-3.90 (m, 1H, CHN), 3.17-3.10 (m, 2H, CH2NHZ), 2.46-2.32 (m, 2H, CH2CHN), 1.90-1.60 (m, 2H, CH2), 1.50-1.25 (m, 4H, CH2), 1.42 (s, 9H, tBu), 1.16 (d, J ) 6.8, 3H, Me). 13C NMR (75 MHz, CDCl3): δ 172.44, 156.90, 136.85, 135.56, 128.89, 128.78, 128.65, 128.37, 67.29, 66.74, 52.01, 44.18, 42.52, 40.41, 31.61, 29.30, 28.46, 22.15, 20.60. FAB-MS: m/z 556 {28, (M + 1)+}, 456 (100). HCl•H-S-β-HAla-Lys(Z)-OBn (13). According to general procedure C, compound 12 (1.00 g, 1.8 mmol) was treated with a saturated HCl/dioxane solution (20 mL). After 30 min, the reaction was completed and the solvent was evaporated. The resulting HCl salt 13 was obtained in almost quantitative yield as a white precipitate (from Et2O) and used in the next coupling step without further purification. Boc-(S-β-HAla)2-Lys(Z)-OBn (14). According to general procedure A, to a solution in DMF (5 mL) of the HCl salt 13 (1 equiv, 1.8 mmol) was added DIEA (0.92 mL, 5.4 mmol). HOBt (304 mg, 2.2 mmol), the acid Boc-β-HAla-OH (1) (365 mg, 1.8 mmol), and then EDC (430 mg, 2.2 mmol) were successively added to the reaction. The residue was purified by recrystallization (CH3Cl/hexane, 20/1) to give compound 14 (850 mg, 74%) as a white solid. 1H NMR (300 MHz, CDCl3): δ 7.38-7.26 (m, 10H ar), 6.68-6.65 (m, 2H, NH), 5.23-5.09 (m, 4H, OCH2Ph, NH), 5.09 (s, 2H, OCH2Ph), 4.58-4.50 (m, 1H, CHN), 4.30-4.20 (m, 1H, CHN), 4.00-3.90 (m, 1H, CHN), 3.18-3.10 (m, 2H, CH2NHZ), 2.42-2.22 (m, 4H, CH2CHN), 1.90-1.62 (m, 2H, CH2), 1.52-1.25 (m, 4H, CH2), 1.42 (s, 9H, tBu), 1.19 (d, J ) 6.5, 3H, Me), 1.15 (d, J ) 6.8, 3H, Me). 13C NMR (75 MHz, CDCl3): δ 172.44, 155.78, 128.88, 128.78, 128.62, 128.37, 67.30, 66.74, 52.27, 43.10, 40.29, 31.29, 28.56, 28.48, 22.21, 20.24. FAB-MS: m/z 663 {22, (M + Na)+}, 641 {38, (M + 1)+}, 541 (100). HCl•H-(S-β-HAla)2-Lys(Z)-OBn (15). According to general procedure C, compound 15 (712 mg, 1.1 mmol) was treated with a saturated HCl/dioxane solution (10 mL). After 30 min, the reaction was completed and the solvent was evaporated. The HCl resulting salt 15 was obtained in almost quantitative yield as a white precipitate (from Et2O) and used in the next coupling step without further purification. Fmoc-Ala-(R-HB)2-OtBu (17). To a solution of the hydroxy derivative 1651 (1 equiv, 4.4 mmol) in CH2Cl2 (40 mL) was added Fmoc-Ala-OH (1.45 g, 4.4 mmol) under argon, and the mixture was cooled to - 5 °C. DCC (0.95 g, 4.62 mmol) and DMAP (0.04 g, 0.22 mmol) were added, and the resulting mixture was allow to warm to room temperature and then stirred for 24 h. The mixture was diluted with Et2O and washed with 1 N HCl, saturated NaHCO3 solution, and brine. The organic layer was dried over anhydrous MgSO4, filtrated, and concentrated. The residue was purified on silica gel (Et2O/

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pentane, 2/3) to give compound 17 (700 mg, 30%) as a white foam. 1H NMR (300 MHz, CDCl3): δ 7.77-7.75 (m, 2H ar), 7.61-7.59 (m, 2H ar), 7.42-7.37 (m, 2H ar), 7.34-7.26 (m, 2H ar), 6.49 (d, J ) 7.2, NH), 5.38-5.22 (m, 2H, CHO), 4.444.32 (m, 3H, CHN, CH2 of Fmoc), 4.25-4.20 (m, 1H, CH of Fmoc), 2.69-2.37 (m, 4H, CH2CHO), 1.43 (s, 9H, tBu), 1.30 (d, J ) 6.2, 3H, Me), 1.29 (d, J ) 6.2, 3H, Me), 1.26 (d, J ) 6.5, 3H, Me). 13C NMR (75 MHz, CDCl3): δ 172.44, 169.69, 169.37, 155.91, 144.23, 144.12, 141.58, 127.94, 125.33, 120.20, 81.10, 68.68, 68.21, 67.09, 49.83, 47.27, 40.05, 40.88, 28.09, 19.80, 19.67, 18.67. FAB-MS: m/z 540 {6, (M + 1)+}, 484 (100). Fmoc-Ala-(R-HB)2-OH (18). According to general procedure C, compound 17 (600 mg, 1.1 mmol) was treated with a TFA/CH2Cl2 (1:1) solution (6 mL). After 30 min, the reaction was completed and the solvent was evaporated. The acid 18 was obtained in almost quantitative yield as a yellow oil and used in the next coupling step without further purification. Fmoc-Ala-(R-HB)2-(S-β-HAla)2-Lys(Z)-OBn (19). According to general procedure A, to a solution in CH2Cl2/DMF (1:1, 12 mL) of the HCl salt 15 (1 equiv, 1.10 mmol) was added DIEA (0.75 mL, 4.40 mmol). HOBt (185 mg, 1.37 mmol), the acid 18 (1 equiv, 1.10 mmol), and then EDC (262 mg, 1.37 mmol) were successively added to the reaction. After 15 h reaction, some of the product precipitated in the reaction mixture. The CH2Cl2 was then evaporated, and the resulting oil was precipitated in a saturated NaHCO3 solution. The precipitate was washed several times with saturated NaHCO3, KHSO4 (1 N) solutions and finally with H2O and dried 15 h under high vacuum. The presence of compound 19 was confirmed by 1H and 13C NMR and MS spectra. Compound 19 was used without further purification. 1H NMR (300 MHz, DMSO-d6): δ 8.28-8.22 (m, 1H, NH), 7.30-7.94 (m, 1H, NH), 7.80-7.74 (m, 1H, NH), 7.75-7.65 (m, 2H ar), 7.42-7.24 (m, 16H ar), 7.22-7.16 (m, 1H, NH), 5.23-5.02 (m, 3H, CHO, NH), 5.208 (s, 2H, OCH2Ph), 5.96 (s, 2H, OCH2Ph), 4.32-4.14 (m, 2H, CHN), 4.10-4.22 (m, 2H, CHN), 2.96-2.87 (m, 2H, CH2NHZ), 2.40-2.00 (m, 8H, CH2CHN, CH2CHO), 1.70-1.51 (m, 2H, CH32), 1.40-1.10 (m, 4H, 2 CH2), 1.23 (d, J ) 6.8, 3H, Me), 1.13 (d, J ) 5.0, 3H, Me), 1.04 (d, J ) 5.9, 3H, Me), 1.030.98 (m, 2 Me). 13C NMR (75 MHz, DMSO-d6): δ 172.28, 170.52, 169.11, 168.09, 167.99, 156.26, 155.99, 144.02, 140.90, 137.43, 136.14, 128.55, 128.47, 128.15, 127.94, 127.84, 127.77, 127.21, 125.38, 120.25, 68.34, 67.77, 65.83, 65.64, 65.12, 63.44, 51.88, 49.42, 46.58, 44.25, 42.18, 42.05, 41.82, 41.64, 30.38, 28.86, 23.18, 22.55, 19.82, 19.75, 19.51, 19.38, 19.15. FABMS: m/z 1028 {23, (M + Na)+}, 1006 {41, (M + 1)+}, 809 (84), 713 (100). H-Ala-(R-HB)2-(S-β-HAla)2-Lys(Z)-OBn (20). The Fmocprotected compound 19 (520 mg, 0.52 mmol) was dissolved in DMF/Et2NH (9:1, 4 mL) under argon and cooled to 0 °C. The mixture was stirred for 1-2 h, and concentration under reduced pressure yielded the crude amine 20 which was identified by NMR and used without further purification. TFA•H-(R-β-HAla)4-Lys(Z)-OBn (22a). According to general procedure B, to a solution in DMF (11 mL) of HCl•H-Lys(Z)-OBn (442 mg, 1.09 mmol) was added DIEA (0.56 mL, 3.27 mmol). HOBt (184 mg, 1.36 mmol), the acid Boc-(β-HAla)4OH (21a)40 (1 equiv, 1.09 mmol), and then EDC (260 mg, 1.36 mmol) were successively added to the reaction. The residue was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-(R-β-HAla)4Lys(Z)-OBn (723 mg, 82% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (3.6 mL), according to the general procedure D, gave the TFA salt 22a, which was used without further purification. TFA•H-(S-β-HAla-R-β-HAla)2-Lys(Z)-OBn (22b). According to general procedure B, to a solution in DMF (2 mL) of HCl•H-Lys(Z)-OBn (62 mg, 0.15 mmol) was added DIEA (0.08 mL, 0.45 mmol). HOBt (26 mg, 0.19 mmol), the acid Boc-(Sβ-HAla-R-β-HAla)2-OH (21b)40 (1 equiv, 0.15 mmol), and then EDC (36 mg, 0.19 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not

soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-(S-β-HAla-R-β-HAla)2-Lys(Z)-OBn (11 mg, 90% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (0.55 mL), according to the general procedure D, gave the TFA salt 22b, which was used without further purification. TFA•H-(S-β-HAla)4-Lys(Z)-OBn (22c). According to general procedure B, to a solution in DMF (15 mL) of HCl•H-Lys(Z)-OBn (515 mg, 1.27 mmol) was added DIEA (0.65 mL, 3.81 mmol). HOBt (214 mg, 1.59 mmol), the acid Boc-(S-β-HAla)4OH (21c)40 (1 equiv, 1.27 mmol), and then EDC (304 mg, 1.59 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc(S-β-HAla)4-Lys(Z)-OBn (819.1 mg, 79% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (4 mL), according to the general procedure D, gave the TFA salt 22c, which was used without further purification. TFA•H-(R-β-HAla)2-(S-β-HAla)2-Lys(Z)-OBn (22d). According to general procedure B, to a solution in DMF (11 mL) of HCl•H-Lys(Z)-OBn (442 mg, 1.09 mmol) was added DIEA (0.56 mL, 3.27 mmol). HOBt (184 mg, 1.36 mmol), the acid Boc-(R-β-HAla)2-(S-β-HAla)2-Lys(Z)-OH (21d)40 (1 equiv, 1.09 mmol), and then EDC (260 mg, 1.36 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-(R-β-HAla)2-(S-β-HAla)2-Lys(Z)-OBn (796 mg, 90% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (3.9 mL), according to the general procedure D, gave the TFA salt 22d, which was used without further purification. TFA•H-Ala-(R-β-HAla)4-Lys(Z)-OBn (23a). According to general procedure B, to a solution in DMF (7 mL) of the TFA salt 22a (1 equiv, 0.89 mmol) was added DIEA (0.61 mL, 3.56 mmol). HOBt (150 mg, 1.11 mmol), Boc-Ala-OH (202 mg, 1.06 mmol), and then EDC (212 mg, 1.1 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-Ala-(R-β-HAla)4-Lys(Z)-OBn (680 mg, 87% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (3.1 mL), according to the general procedure D, gave the TFA salt 23a, which was used without further purification. TFA•H-Ala-(S-β-HAla-R-β-HAla)2-Lys(Z)-OBn (23b). According to general procedure B, to a solution in DMF (2 mL) of the TFA salt 22b (1 equiv, 0.135 mmol) was added DIEA (0.092 mL, 0.540 mmol). HOBt (23 mg, 0.169 mmol), Boc-AlaOH (31 mg, 0.162 mmol), and then EDC (32 mg, 0.169 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired BocAla-(S-β-HAla-R-β-HAla)2-Lys(Z)-OBn (73 mg, 61% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (0.33 mL), according to the general procedure D, gave the TFA salt 23b, which was used without further purification. TFA•H-Ala-(S-β-HAla)4-Lys(Z)-OBn (23c). According to general procedure B, to a solution in DMF (10 mL) of the TFA salt 22c (1 equiv, 1.00 mmol) was added DIEA (0.68 mL, 4.00 mmol). HOBt (169 mg, 1.25 mmol), Boc-Ala-OH (227 mg, 1.20 mmol), and then EDC (239 mg, 1.25 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-Ala-(S-β-HAla)4-Lys(Z)-OBn (762 mg, 86% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (3.5 mL), according to the general procedure D, gave the TFA salt 23c, which was used without further purification. TFA•H-Ala-(R-β-HAla)2-(S-β-HAla)2-Lys(Z)-OBn (23d).

Nonapeptide Analogue Binding Affinity to MHC Protein

Journal of Medicinal Chemistry, 1999, Vol. 42, No. 13 2327

According to general procedure B, to a solution in DMF (10 mL) of the TFA salt 22d (770 mg, 0.93 mmol) was added DIEA (0.64 mL, 3.72 mmol). HOBt (157 mg, 1.16 mmol), Boc-AlaOH (211 mg, 1.12 mmol), and then EDC (215 mg, 1.16 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired BocAla-(R-β-HAla)2-(S-β-HAla)2-Lys(Z)-OBn (490 mg, 60% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (2.2 mL), according to the general procedure D, gave the TFA salt 23d, which was used without further purification. TFA•H-Arg(NO2)-Ala-(S-β-HAla-R-HB)2-Lys(Z)-OBn (24). According to general procedure A, to a solution in CH2Cl2/DMF (4:3, 8 mL) of the TFA salt 11 (1 equiv, 0.74 mmol) was added DIEA (0.38 mL, 2.22 mmol). HOBt (125 mg, 0.92 mmol), BocArg(NO2)-OH (259 mg, 0.81 mmol), and then EDC (176 mg, 0.92 mmol) were successively added to the reaction. The resulting residue was purified on silica gel (CH2Cl2/MeOH, 9/1) to give compound Boc-Arg(NO2)-Ala-(S-β-HAla-R-HB)2-Lys(Z)OBn (650 mg, 82%) as a fine white powder. 1H NMR (300 MHz, CDCl3): δ 8.45-8.30 (m, 1H, NH), 7.80-7.60 (m, 3H, NH), 7.8-7.28 (m, 10H ar), 7.20-7.12 (m, 3H, NH), 5.70-5.60 (m, 1H, NH), 5.34-5.26 (m, 1H, NH), 5.26-5.06 (m, 4H, CHO, OCH2Ph), 5.07 (s, 2H, OCH2Ph), 4.60-4.50 (m, 1H, CHN), 4.46-4.2 (m, 4H, CHN), 3.36-3.24 (m, 2H, CH2NHC), 3.163.07 (m, 2H, CH2NHZ), 2.53-2.33 (m, 8H, CH2CHN, CH2CHO), 1.90-1.60 (m, 6H, CH2), 1.54-1.10 (m, 4H, CH2) 1.41 (s, 9H, tBu), 1.34 (d, J ) 6.8, 3H, Me), 1.28 (d, J ) 6.2, 3H, Me), 1.24 (d, J ) 6.5, 3H, Me), 1.20-1.16 (m, 6H, Me). 13C NMR (75 MHz, CDCl3): δ 172.61, 170.35, 170.27, 169.77, 157.09, 136.82, 135.61, 128.87, 128.80, 128.54, 128.37, 128.23, 80.46, 68.68, 68.40, 67.22, 65.96, 52.48, 49.54, 42.48, 42.16, 40.46, 31.36, 29.41, 28.39, 24.85, 22.41, 20.02, 19.88, 19.74, 19.66, 18.19. FAB-MS: m/z 1107 {60, (M + Na)+}, 1085 {100, (M + 1)+}, 985 (22), 713 (7). Further treatment with TFA (5 mL), according to the general procedure D, gave the TFA salt 24, which was used without further purification. TFA•H-Arg(NO2)-Ala-(R-HB)2-(S-β-HAla)2-Lys(Z)-OBn (25). According to general procedure B, to a solution in DMF (6 mL) of the TFA salt 20 (1 equiv, 0.52 mmol) was added DIEA (0.26 mL, 1.55 mmol). HOBt (87 mg, 0.65 mmol), BocArg(NO2)-OH (198 mg, 0.62 mmol), and then EDC (123 mg, 0.65 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-Arg(NO2)-Ala-(R-HB)2-(S-β-HAla)2-Lys(Z)-OBn (409 mg, 77% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (1.4 mL), according to the general procedure D, gave the TFA salt 25, which was used without further purification. TFA•H-Arg(NO2)-Ala-(R-β-HAla)4-Lys(Z)-OBn (26a). According to general procedure B, to a solution in DMF (9 mL) of the TFA salt 23a (1 equiv, 0.77 mmol) was added DIEA (0.53 mL, 3.08 mmol). HOBt (130 mg, 0.96 mmol), Boc-Arg(NO2)OH (295 mg, 0.92 mmol) and then EDC (183 mg, 0.96 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any solvent to be purified. The presence of the desired Boc-Arg(NO2)-Ala-(R-βHAla)4-Lys(Z)-OBn (757 mg, 81% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (2.7 mL), according to the general procedure D, gave the TFA salt 26a, which was used without further purification. TFA•H-Arg(NO2)-(S-β-HAla-R-β-HAla)2-Lys(Z)-OBn (26b). According to general procedure B, to a solution in DMF (9 mL) of the TFA salt 23b (1 equiv, 0.83 mmol) was added DIEA (0.057 mL, 0.33 mmol). HOBt (14 mg, 0.104 mmol), Boc-Arg(NO2)-OH (32 mg, 0.099 mmol), and then EDC (20 mg, 0.104 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chroma-

tography or HPLC solvent. The presence of the desired BocArg(NO2)-(S-β-HAla-R-β-HAla)2-Lys(Z)-OBn (74 mg) was confirmed by FAB-MS. Further treatment with TFA (0.5 mL), according to the general procedure D, gave the TFA salt 26b, which was used without further purification. TFA•H-Arg(NO2)-Ala-(S-β-HAla)4-Lys(Z)-OBn (26c). According to general procedure B, to a solution in DMF (9 mL) of the TFA salt 23c (1 equiv, 0.86 mmol) was added DIEA (0.59 mL, 3.44 mmol). HOBt (145 mg, 1.07 mmol), Boc-Arg(NO2)OH (329 mg, 1.03 mmol), and then EDC (205 mg, 1.07 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired BocArg(NO2)-Ala-(S-β-HAla)4-Lys(Z)-OBn (860 mg, 82% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (3.1 mL), according to the general procedure D, gave the TFA salt 26c, which was used without further purification. TFA•H-Arg(NO2)-Ala-(R-β-HAla)2-(S-β-HAla)2-Lys(Z)OBn (26d). According to general procedure B, to a solution in DMF (9 mL) of the TFA salt 23d (1 equiv, 0.45 mmol) was added DIEA (0.31 mL, 1.81 mmol). HOBt (76 mg, 0.56 mmol), Boc-Arg(NO2)-OH (172 mg, 0.54 mmol), and then EDC (107 mg, 0.56 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-Arg(NO2)-Ala-(R-β-HAla)2-(S-β-HAla)2-Lys(Z)-OBn (460 mg, 84% crude) was confirmed by 1H and 13C NMR and MS spectra. Further treatment with TFA (2.2 mL), according to the general procedure D, gave the TFA salt 26d, which was used without further purification. H-Gly-Arg-Ala-(S-β-HAla-R-HB)2-Lys-OH (27). According to general procedure B, to a solution in DMF (6 mL) of the TFA salt 24 (1 equiv, 0.50 mmol) was added DIEA (0.26 mL, 1.51 mmol). HOBt (85 mg, 0.63 mmol), Boc-Gly-OH (97 mg, 0.55 mmol), and then EDC (124 mg, 0.63 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any solvent to be purified. The presence of the desired Boc-Gly-Arg(NO2)-Ala-(S-β-HAla-R-HB)2-Lys(Z)-OBn (447 mg, 68% crude) as a fine yellow-white powder was confirmed by 1H and 13C NMR and MS spectra. According to general procedure E, Boc-Gly-Arg(NO2)-Ala(S-β-HAla-R-HB)2-Lys(Z)-OBn (300 mg, 0.26 mmol) was dissolved in TFE/CH3COOH (3:1, 4 mL) and hydrogenated in the presence of Pd/BaSO4 (10%, 60 mg). The resulting precipitate was purified by HPLC C8 (5-40% B, 30 min), tR 12.5 min, to give after lyophilization the pure compound 27 in about 40% yield. 1H NMR (300 MHz, D2O): δ 5.24-5.10 (m, 2H, CHO), 4.34-4.28 (m, 2H, CHN), 4.22-4.15 (m, 3H, CHN), 3.84 (s, 2H, CH2N), 3.19 (t, J ) 6.8, 2H, CH2NHC), 3.00-2.95 (m, 2H, CH2NH2), 2.58-2.42 (m, 8H, CH2CHO, CH2CHN), 1.92-1.58 (m, 8H, CH2), 1.50-1.40 (m, 2H, CH2), 1.32 (d, J ) 7.5, 3H, Me), 1.26 (d, J ) 6.2, 3H, Me), 1.25 (d, J ) 6.2, 3H, Me), 1.181.13 (m, 6H, Me). FAB-MS: m/z 811 {10, (M + K)+}, 795 {32, (M + Na)+}, 773 {100, (M + 1)+}. Purity by analytical HPLC (0-100% B, 60 min, tR 18.7 min) >99%. H-Gly-Arg-Ala-(R-HB)2-(S-β-HAla)2-Lys-OH (28). According to general procedure D, to a solution in DMF (5 mL) of the TFA salt 25 (1 equiv, 0.37 mmol) was added DIEA (0.19 mL, 1.11 mmol). HOBt (62 mg, 0.46 mmol), Boc-Gly-OH (77 mg, 0.44 mmol), and then EDC (88 mg, 0.46 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-Gly-Arg(NO2)-Ala-(R-HB)2-(S-β-HAla)2-Lys(Z)-OBn (252 mg, 60% crude) was confirmed by 1H and 13C NMR and MS spectra. According to general procedure E, Boc-Gly-Arg(NO2)-Ala(R-HB)2-(S-β-HAla)2-Lys(Z)-OBn (200 mg, 0.17 mmol) was dissolved in TFE/CH3COOH (3:1, 3.5 mL) and hydrogenated in the presence of Pd/BaSO4 (10%, 40 mg). The resulting

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Poenaru et al.

precipitate was purified by HPLC (10-40% B, 30 min), tR 6.2 min, to give after lyophilization the pure compound 28 in about 25% yield. 1H NMR (300 MHz, D2O): δ 5.22-5.06 (m, 2H, CHO), 4.28-4.20 (m, 3H, CHN), 4.16-4.04 (m, 2H, CHN), 3.76 (s, 2H, CH2N), 3.16-3.10 (m, 2H, CH2NHC), 2.93-2.86 (m, 2H, CH2NH2), 2.65-2.49 (m, 2H, CH2CHO), 2.43-2.32 (m, 2H, CH2CHO), 2.36 (d, J ) 7.2, 2H, CH2CHN), 2.26 (d, J ) 7.2, 2H, CH2CHN), 1.85-1.53 (m, 8H, CH2), 1.41-1.28 (m, 2H, CH2), 1.35 (d, J ) 7.2, 3H, Me), 1.20-1.14 (m, 6H, Me), 1.081.03 (m, 6H, Me). FAB-MS: m/z 811 {12, (M + K)+}, 795 {23, (M + Na)+}, 773 {100, (M + 1)+}. Purity by analytical HPLC (0-100% B, 60 min, tR 19.5 min) >99%. H-Gly-Arg-Ala-(R-β-HAla)4-Lys-OH (29a). According to general procedure B, to a solution in DMF (7 mL) of the TFA salt 26a (1 equiv, 0.68 mmol) was added DIEA (0.47 mL, 2.71 mmol). HOBt (115 mg, 0.85 mmol), Boc-Gly-OH (143 mg, 0.82 mmol), and then EDC (163 mg, 0.85 mmol) were successively added to the reaction. The precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-Gly-Arg(NO2)-Ala-(R-β-HAla)4-Lys(Z)-OBn (622 mg, 80% crude) was confirmed by 1H and 13C NMR and MS spectra. According to general procedure E, Boc-Gly-Arg(NO2)-Ala(R-β-HAla)4-Lys(Z)-OBn (200 mg, 0.18 mmol) was dissolved in TFE/CH3COOH (3:1, 3.5 mL) and hydrogenated in the presence of Pd/BaSO4 (10%, 40 mg). The resulting precipitate was purified by HPLC (5-40% B, 30 min), tR 17.20 min, to give after lyophilization the pure compound 29a in about 25% yield. 1H NMR (300 MHz, D2O): δ 4.32-4.24 (m, 2H, CHN), 4.21-4.07 (m, 5H, CHN), 3.85-3.80 (m, 2H, CH2N), 3.20-3.14 (m, 2H, CH2NHC), 2.98-2.92 (m, 2H, CH2NH2), 2.50-2.25 (m, 8H, CH2CHN), 1.90-1.58 (m, 8H, CH2), 1.47-1.36 (m, 2H, CH2), 1.32 (d, J ) 7.2, 3H, Me), 1.14-1.09 (m, 12H, Me). 13C NMR (75 MHz, D2O): δ 178.57, 176.08, 175.29, 56.37, 55.39, 52.77, 46.22, 46.11, 45.20, 43.36, 43.15, 41.95, 32.66, 31.13, 28.98, 27.05, 24.86, 22.00, 19.43. FAB-MS: m/z 1542 {9, (2M +2)+}, 771 {100, (M + 1)+}. Purity by analytical HPLC (0100% B, 60 min, tR 16.4 min) >99%. H-Gly-Arg-Ala-(S-β-HAla-R-β-HAla)2-Lys-OH (29b). According to general procedure B, to a solution in DMF (2 mL) of the TFA salt 26b (1 equiv, 0.068 mmol) was added DIEA (0.046 mL, 0.28 mmol). HOBt (12 mg, 0.085 mmol), Boc-GlyOH (14 mg, 0.082 mmol), and then EDC (16 mg, 0.085 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired BocGly-Arg(NO2)-(S-β-HAla-R-β-HAla)2-Lys(Z)-OBn (70 mg) was confirmed by FAB-MS. According to general procedure E, Boc-Gly-Arg(NO2)-(S-βHAla-R-β-HAla)2-Lys(Z)-OBn (70 mg, 0.06 mmol) was dissolved in TFE/CH3COOH (3:1, 1 mL) and hydrogenated in the presence of Pd/BaSO4 (10%, 10 mg). The resulting precipitate was purified by HPLC (5-40% A, 30 min), tR 8.6 min, to give after lyophilization the pure compound 29b in about 25% yield. 1H NMR (300 MHz, D O): δ 4.26-4.18 (m, 2H, CHN), 4.182 4.04 (m, 5H, CHN), 3.76-3.73 (m, 2H, CH2N), 3.12-3.07 (m, 2H, CH2NHC), 2.91-2.85 (m, 2H, CH2NH2), 2.40-2.32 (m, 2H, CH2CHN), 2.30-2.22 (m, 6H, CH2CHN), 1.85-1.50 (m, 8H, CH2), 1.40-1.30 (m, 2H, CH2), 1.26-1.22 (m, 3H, Me), 1.091.02 (m, 12H, Me). FAB-MS: m/z 771 (86, [M + 1]+). Purity by analytical HPLC (0-100% B, 60 min, tR 15.4 min) >99%. H-Gly-Arg-Ala-(S-β-HAla)4-Lys-OH (29c). According to general procedure B, to a solution in DMF (8 mL) of the TFA salt 26c (1 equiv, 0.77 mmol) was added DIEA (0.53 mL, 3.1 mmol). HOBt (131 mg, 0.97 mmol), Boc-Gly-OH (163 mg, 0.93 mmol), and then EDC (185 mg, 0.97 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-Gly-Arg(NO2)-Ala-(S-β-HAla)4Lys(Z)-OBn (719 mg, 82% crude) was confirmed by 1H and 13C NMR and MS spectra.

According to general procedure E, Boc-Gly-Arg(NO2)-Ala(S-β-HAla)4-Lys(Z)-OBn (340 mg, 0.30 mmol) was dissolved in TFE/CH3COOH (3:1, 4 mL) and hydrogenated in the presence of Pd/BaSO4 (10%, 60 mg). The resulting precipitate was purified by HPLC (10-40% B, 30 min), tR 6.8 min, to give after lyophilization the pure compound 29c in about 30% yield. 1H NMR (300 MHz, D2O): δ 4.34-4.27 (m, 2H, CHN), 4.24-4.09 (m, 5H, CHN), 3.84-3.81 (m, 2H, CH2N), 3.21-3.15 (m, 2H, CH2NHC), 2.99-2.93 (m, 2H, CH2NH2), 2.45-2.41 (m, 2H, CH2C9HN), 2.40-2.26 (m, 6H, CH2CHN), 1.92-1.57 (m, 8H, CH2), 1.47-1.35 (m, 2H, CH2), 1.31 (d, J ) 7.2, 3H, Me), 1.151.09 (m, 12H, Me). 13C NMR (75 MHz, D2O): δ 178.59, 176.58, 176.50, 176.08, 175.30, 170.09, 159.82, 56.22, 55.30, 52.71, 46.25, 45.22, 44.78, 43.37, 43.13, 42.00, 32.78, 31.13, 28.99, 27.02, 24.85, 22.16, 21.97, 19.50. FAB-MS: m/z 1542 {17, (2M + 2)+}, 771 {100, (M + 1)+}. Purity by analytical HPLC (0100% B, 60 min, tR 15.3 min) >99%. H-Gly-Arg-Ala-(R-β-HAla)2-(S-β-HAla)2-Lys-OH (29d). According to general procedure B, to a solution in DMF (5 mL) of the TFA salt 26d (1 equiv, 0.42 mmol) was added DIEA (0.29 mL, 1.68 mmol. HOBt (71 mg, 0.52 mmol), Boc-Gly-OH (88 mg, 0.50 mmol), and then EDC (100 mg, 0.52 mmol) were successively added to the reaction. The resulting precipitate was dried under high vacuum and used without further purification as it is not soluble in any flash chromatography or HPLC solvent. The presence of the desired Boc-Gly-Arg(NO2)-Ala-(R-β-HAla)2-(S-β-HAla)2-Lys(Z)-OBn (333 mg, 70% crude) was confirmed by 1H and 13C NMR and MS spectra. According to general procedure E, Boc-Gly-Arg(NO2)-Ala(R-β-HAla)2-(S-β-HAla)2-Lys(Z)-OBn (150 mg, 0.13 mmol) was dissolved in TFE/CH3COOH (3:1, 3 mL) and hydrogenated in the presence of Pd/BaSO4 (10%, 25 mg). The resulting precipitate was purified by HPLC (2-40% B, 30 min), tR 20.5 min, to give after lyophilization the pure compound 29d in about 20% yield. 1H NMR (300 MHz, D2O): δ 4.36-4.28 (m, 2H, CHN), 4.27-4.12 (m, 5H, CHN), 3.87-3.83 (m, 2H, CH2N), 3.23-3.18 (m, 2H, CH2NHC), 3.01-2.96 (m, 2H, CH2NH2), 2.346-2.43 (m, 2H, CH2CHN), 2.41-2.31 (m, 6H, CH2CHN), 1.92-1.60 (m, 8H, CH2), 1.50-1.40 (m, 2H, CH2), 1.35 (d, J ) 7.5, 3H, Me), 1.17-1.11 (m, 12H, Me). FAB-MS: m/z 793 {15, (M + Na)+}, 771 {100, (M + 1)+}. Purity by analytical HPLC (0-100% B, 60 min, tR 15.4 min) >80%. Molecular Dynamics Simulations. Molecular mechanics and dynamics calculations were realized using the AMBER 5.0 package66 using the parm96 parameter set and an all-atom force-field representation.67 Force-field parameters for the ester bonds were taken from the literature.68 Atomic charges for the new monomers (R-HB, S-β-HAla, R-β-HAla) were calculated using the GAUSSIAN94 package69 and the HF/6-31G* basis set, by fitting atom-centered charges to an ab initio electrostatic potential, using the RESP method.70 Initial coordinates for the MHC-ligand complexes were obtained from the X-ray structure of HLA-B*270556 (Protein Data Bank code 1hsa) as previously described.33,34 The spacers were substituted for the natural pentapeptide sequence using the SYBYL 6.3 modeling package (TRIPOS Assoc., Inc., St. Louis, MO). From a starting fully extended conformation, dihedral angles of the main chain between P3 and P9 were modified in order to reproduce a correct trans geometry for the newly introduced amide or ester bonds. The ligand was first relaxed by 1000 steps of conjugate gradient energy minimization while maintaining the protein fixed. It was then submitted to a 100-ps Simulated annealing (SA) protocol in order to sample the broadest possible conformational space. Starting with random velocities assigned at a temperature of 1000 K, the peptide was first coupled to a heat bath at 1000 K using a temperature coupling constant Tτ of 0.2 ps and then linearly cooled to 50 K for the next 50 ps while strengthening Tτ to a value of 0.05 ps. During these 100 ps, no protein atom was allowed to move. As the simulated annealing was performed in vacuo, a distance-dependent dielectric function (
) 4r) was used. A twin cutoff (10.0, 15.0 Å) was used to calculate nonbonded electrostatic interactions at every minimization step and every nonbonded pair list update (10 steps), respectively.

Nonapeptide Analogue Binding Affinity to MHC Protein From the last SA conformer, 13 counterions (9 Na+ and 4 Cl- ions) were then placed at electrostatic minima to neutralize the protein, using the CION routine of AMBER.66 It was then solvated in a 10-Å thick TIP3P water shell. After the solvent was minimized by 1000 steps of steepest descent, the solvent (water and counterions) was equilibrated by 25-ps MD at 300 K. The solvent was minimized again, and the fully solvated complex was finally relaxed by 1000 steps of steepest descent. The obtained coordinates were then used as a starting point for a 500-ps MD simulation at 300 K. To avoid large drifts from the protein crystal structure, a weak positional harmonic constraint of 0.05 kcal‚mol-1‚Å-1 was applied to backbone atoms of B*2705. As the solvent was implicitly taken into account, a constant dielectric function (
) 1) was utilized. For the whole trajectory, the same twin cutoff (10-15 Å) was used for calculating nonbonded interactions, and the nonbonded pair list was updated every 10 steps. The SHAKE algorithm was used on hydrogens with a tolerance of 0.00025 Å, a time step of 2 fs, and Berendsen temperature coupling with separate coupling of solute and solvent atoms to the heat bath. Coordinates, velocities, and energies were saved every 0.5 ps. All computations were done using the parallel version of AMBER5.0 implemented on a CRAY J90 cluster and an INTEL paragon machine. The analyses of molecular dynamics trajectories were achieved using in-house routines and the CARNAL module of AMBER.66 Epitope Stabilization Assay. The quantitative assay used was previously described.71 Briefly, RMA-S transfectants expressing B*2705 were used. These are murine cells with impaired TAP-mediated peptide transport and low surface expression of (empty) class I MHC molecules, which can be induced at 26 °C72 and stabilized at the cell surface through binding of exogenously added ligands. These cells were incubated at 26 °C for 24 h. After this time they were incubated for 1 h at 26 °C with 10-4-10-9 M peptides, transferred to 37 °C, and collected after 4 h for flow microcytometry (FMC) analysis with the ME1 mAb (IgG1, specific for HLA-B27, -B7, and -B22).73 The determinant recognized by ME1 is not affected by bound peptides (data not shown). Binding of a given ligand was measured as its C50. This is its molar concentration at 50% of the fluorescence obtained with that ligand at 10-4 M. Ligands with C50 e 5 µM were considered to bind with high affinity, as these were the values obtained for most of the natural B27-bound peptides. C50 values between 5 and 50 µM were considered to reflect intermediate affinity. C50 g 50 µM indicated low affinity.

Acknowledgment. This work is supported by the Schweizerischer Nationalfonds zur Fo¨rderung der Wissenschaftlichen Forschung (Project No. 31-45504.95) and by Grant SAF97/0182 from the Spanish Plan Nacional de I+D to J.A.L.C. D.S. thanks Novartis Pharma (Basel) for continuous financial support to his group and A. K. Beck, J. Schreiber, and S. Sigrist for processing the manuscript. D.R. thanks the calculation center of the ETH-Zu¨rich for allocation of computer time on the CRAY J90 and PARAGON supercomputers. References (1) Heemels, M. T.; Ploegh, H. L. Generation, translocation and presentation of MHC class I-restricted peptides. Annu. Rev. Biochem. 1995, 64, 643-691. (2) Batalia, M. A.; Collins, E. J. Peptide binding by class I and class II MHC molecules. Biopolymers 1997, 43, 281-302. (3) Falk, K.; Ro¨tzschke, O.; Stevanovic, S.; Jung, G.; Rammensee, H.-G. Allele-specific motifs revealed by sequencing of selfpeptides eluted from MHC molecules. Nature 1991, 351, 290296. (4) Rammensee, H. G.; Friede, T.; Stevanovic, S. MHC ligands and peptide motifs: first listing. Immunogenetics 1995, 41, 178-228. (5) Saper, M. A.; Bjorkman, P. J.; Wiley, D. C. Refined structure of the human histocompatibility antigen HLA-A2 at 2.6 Å resolution. J. Mol. Biol. 1991, 219, 277-319.

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