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Molecular Mimicry in Innate Immunity:
Structure of a Bacterial TIRVirulence Factor
Jaime Pascual, Burnham Institute
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Suzuki, Trends in Immunology (2002)
Toll-like receptor signaling pathway
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Toll-like receptor activation mechanismDeGrado, Structure (2008)
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TIR 3D fold belongsto the flavodoxin-like
/ familythat includes
bacterial proteins
Crystal structure of monomeric humanToll-like receptor 1 TIR domain
PDB_ID: 1FYV
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Model for TIR-TIR signaling interaction:homotypic via E-helix and heterotypic via BB loop
Jiang, PNAS (2006)
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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)
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Sequence alignment between prokaryotic & eukaryotic TIRs
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Native protein crystal of P. denitrificans TLP TIR domain
Crystallization conditions at 4 C:
Salt: 0.2 M Ammonium sulfate
Buffer: 0.1 M Sodium cacodylate pH 6.0
Precipitant: 31% (w/v) PEG 8000
Dataset collected in-house:
Resolution: 2.50
Space group: P 2 12121
Unit cell: a=80.8; b=85.6; c=89.6
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Pd TLP TIR structure has a TIR foldSuperposition of
Pd
TLP-TIRdomain onto human TLR1-TIRdomain (C RMSD = 2.8 )
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Chain A
Chain B
BB loop
BB loop
DD loop
EE loop
Pd TLP TIR crystal dimer :Exposed BB loops available for hetero typic interactions
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0
100
200
300
400
500
600
0 20 40 60 80 100
m A U
ml
-80
-70
-60
-50
-40
-30
-20
-10
0
0 20 40 60 80 100
m A U
ml
-100
0
100
200
300
400
500
600
0 20 40 60 80 100
m A U
ml
158kDa 44kDa 17kDa
Gel filtrationchromatograms for Pd TLP
Full length(33 kDa)
C-terminalTIR(17 kDa)
N-terminalHelical bundle(16 kDa)
Dimer
Monomer
Oligomer
aa299
TIRHelical bundlepI 5.7pI 10.2 Full length pI 9.6
Leu145aa1 aa299
TIRHelical bundlepI 5.7pI 10.2 Full length pI 9.6
Leu145aa1 aa299
TIRHelical bundlepI 5.7pI 10.2 Full length pI 9.6
Leu145aa1
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Dimerization Interface Determined UsingDeuterium Exchange Mass Spectrometry ( DXMS)
Pd TIR - monomer
full lengthPd TLP -
dimer
HB
HB
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DXMS of Pd TIR vs Pd TLP:Same protected interface in Pd TLP as in the Pd TIR crystal dimer
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DXMS of Pd TIR vs Pd TLP:Same protected interface in Pd TLP as in the Pd TIR crystal dimer
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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 increasedlevels of pro-inflammatory cytokine secretion
Escherichia coli (Cirl, et al., 2008) :TIR-like virulence factor directly interacts with human MyD88
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Drugabilityof the TLR pathway
Beutler, Nature (2004)
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BB loop mimic as inhibitor of prot-prot inter:a low molecular weight pyrrolidine interacts with MyD88TIR
Rebek & Bartfai, PNAS (2006)
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L R R
D e a t
h
D o m a i n
MyD88
NF-B activation NF-B activation
PAMP
TLR
TIR
Healthy macrophage Infected macrophage
TIR virulence factor
H e
l i c a
l
b u n
d l
e
TIR
Model for virulence action by TIR mimicry
IRAKkinase
MyD88
TLR
PAMP
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Acknowle dgements
Siew Leong Chan & Lieh Yoon Low
Eugenio Santelli (Robert C.Liddington)
Gaelle Le Negrate (John C. Reed)
Group
Collaborators
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