Tir Skistone Talk

Molecular Mimicry in Innate Immunity:

Structure of a Bacteria TIR Virulence Factor Jaime Pascual, Burnham Institute

Toll-like receptor signaling pathway

κ Suzuki, Trends in Immunology (2002)

Crystal structure of monomeric human Toll-like receptor 1 TIR domain

PDB_ID: 1FYV

TIR 3D fold belongs to the flavodoxin-like α/β family that includes bacterial proteins

Model for TIR-TIR signaling interaction: homotypic via E-helix and heterotypic via BB loop

Jiang, PNAS (2006)

Sequence alignment between prokaryotic & eukaryotic TIRs

Gel filtration chromatograms for PdTLP aa1

Leu145

Coiled­coil pI 10.2

600

Dimer

500

TIR­like

Full length pI 9.6 158kDa

44kDa

pI 5.7

17kDa

mAU

400

Full length (33 kDa)

300 200 100 0 0

20

40

60

80

100

ml 0 -10

Oligomer

-20

N-terminal Coiled-coil (16 kDa)

mAU

-30 -40 -50 -60 -70 -80 0

20

40

60

80

100

ml

600

Monomer

500

C-terminal TIR (17 kDa)

mAU

400 300 200 100 0 -100 0

20

40

60 ml

80

100

aa299

Native protein crystal of PdTLP TIR domain Crystallization conditions at 4° C:

Dataset collected in-house:

Salt: 0.2 M Ammonium sulfate

Resolution: 2.75 Å

Buffer: 0.1 M Sodium cacodylate pH 6.5

Space group: P 212121

Precipitant: 30% (w/v) PEG 8000

Unit cell: a=80.5; b=85.6; c=90.1

PdTLP TIR structure is a TIR fold Superposition of P.denitrificans TLP-TIR domain onto human TLR1-TIR domain (Cα RMSD = 2.8 Å)

PdTLP TIR crystal dimer: Exposed BB loops available for heterotypic interactions Chain B DD loop

BB loop EE loop Chain A

BB loop

DXMS of PdTIR vs PdTLP : Same protected interface as in the PdTIR crystal dimer

2

Sm 4

2

ad be

ac

88 M yD 4

se p

em

M.W. (kDa)

p

vc

t

Co-immunoprecipitation between MyD88 and PdTLP TIR domains *

37

IP-Myc IB-HA

26

* *

19

IP-Myc IB-Myc

15

37

*

Lysate IB-HA 26

Model for virulence action by TIR mimicry Healthy macrophage

Infected macrophage

PAMP

TLR

LRR

TLR

PAMP

IRAK

kinase

Coiled-coil

MyD88

Death Domain

TIR

TIR virulence factor

TIR

MyD88

NF-кB activation

NF-кB activation

Acknowledgements Group

Siew Leong Chan & Ray Low Collaborators

Eugenio Santelli (Robert C. Liddington) Gaelle Le Negrate (John C. Reed)