Molecular Mimicry In Innate Immunity:: Structure Of A Bacterial Tir Virulence Factor

Molecular Mimicry in Innate Immunity:

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

Toll-like receptor signaling pathway

κ Suzuki, Trends in Immunology (2002)

Toll-like receptor activation mechanism DeGrado, Structure (2008)

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)

Striking a balance through host mimicry: Myxoma virus M11L virulence factor: a Bcl-2 mimic binding and antagonizing pro-apoptotic Bax & Bak

Colman, Mol Cell (2007)

Sequence alignment between prokaryotic & eukaryotic TIRs

Native protein crystal of P. denitrificans TLP TIR domain Crystallization conditions at 4° C:

Dataset collected in-house:

Salt: 0.2 M Ammonium sulfate

Resolution: 2.50 Å

Buffer: 0.1 M Sodium cacodylate pH 6.0

Space group: P 212121

Precipitant: 31% (w/v) PEG 8000

Unit cell: a=80.8; b=85.6; c=89.6

PdTLP TIR structure has a TIR fold Superposition of PdTLP-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

Gel filtration chromatograms for PdTLP aa1

Leu145

Helical bundle pI 10.2

600

Dimer

500

TIR

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 Helical bundle (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

Dimerization Interface Determined Using Deuterium Exchange Mass Spectrometry (DXMS)

PdTIR monomer

H B

H B

full length PdTLP dimer

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

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

4µg

2µg

be a ph Se

2µg

Sm

M 4µg

ac

88 yD

t vc p Em

M.W. (kDa)

ds

Co-immunoprecipitation between MyD88 and PdTLP TIR domains *

37

IP-Myc IB-HA

26

*

MyD88

* 19

IP-Myc IB-Myc

PdTLP 15 37

MyD88 Smac

26

*

Lysate IB-HA

Functional studies on bacteria TLP proteins Salmonella enterica (Newman, et al., 2006) : delayed disease effects of a TIR-like knockout strain infecting mice

Brucella abortus (Salcedo, et al., 2008) : a TIR-like knockout strain infecting human dendritic cells produced increased levels of pro-inflammatory cytokine secretion

Escherichia coli (Cirl, et al., 2008) : TIR-like virulence factor directly interacts with human MyD88

Drugability of the TLR pathway

Beutler, Nature (2004)

BB loop mimic as inhibitor of prot-prot inter: a low molecular weight pyrrolidine interacts with MyD88 TIR

Rebek & Bartfai, PNAS (2006)

Model for virulence action by TIR mimicry Healthy macrophage

Infected macrophage

PAMP

TLR

LRR

TLR

PAMP

IRAK

kinase

Helical bundle

MyD88

Death Domain

TIR

TIR virulence factor

TIR

MyD88

NF-кB activation

NF-кB activation

Acknowledgements Group

Siew Leong Chan & Lieh Yoon Low Collaborators

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