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Structural mimicry in virulence factors:

Bacteria TIR-like proteins

Jaime Pascual, Burnham Institute



Toll-like receptor activation mechanismDeGrado, Structure (2008)



TIR 3D fold belongsto the flavodoxin-likeα/β family

that includes

bacterial proteins

Crystal structure of monomeric humanToll-like receptor 1 TIR domain

PDB_ID: 1FYV



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

Jiang, PNAS (2006)



Sequence alignment between prokaryotic & eukaryotic TIRs



Genome location of TIR-like genes:

pathogenicity islands of infectious bacteria

(1) TIR-like gene; (20) integrase gene; (12) type IV secretion pilin protein precursor gene



Prokaryotic TIRs Status

Bacillus subtilis Cloned Brucella abortus Cloned, no expression

Burkholderia fungorum Cloned, no expression

Escherichia coli CFT073 Cloned, toxic, refolded, unstable

Paracoccus denitrificans Cloned, expressed, purified, >20mg/ml, xtal trials

Salmonella enteriditis Cloned, toxicStaphylococus aureus Cloned, no expression

Prokaryote TIR-like proteinsexpression trials in E. coli

P. denitrificans TIR-like protein (Pd TLP)

could only be expressed in high quantity

using the Rosetta(DE3)-pLysS E. coli strain.



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158kDa 44kDa 17kDa

Gel filtration chromatograms for Pd TLP

Full length(33 kDa)

C-terminalTIR-like(17 kDa)

N-terminalCoiled-coil(16 kDa)

Dimer

Monomer

Oligomer

TIR-likeCoiled-coilpI 5.7pI 10.2 Full length pI 9.6

Leu145 aa299aa1



Differential scanning calorimetry profiles forPd TLP full length, coiled-coil & TIR-like domains



1H-15N 2D HSQC NMR spectrum of

Pd TLP TIR-like domain

( p p m )

(ppm)



Pd TLP TIR-like domain is a TIRSuperposition of P. denitrificans Tlp-TIR domain onto human TLR1-TIR domain

(Cα RMSD = 2.8 Å)



Pd TLP TIR-like crystal dimer:

Exposed BB loops available for heterotypic interactions



Functional studies on bacteria TLP proteins

Salmonella enterica (Newman, et al., 2006) :

delayed 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



SalmonellaSalmonellaSalmonellaSalmonella ParacoccusParacoccusParacoccusParacoccus BrucellaBrucellaBrucellaBrucella

P. denitrificans TIR-like protein suppress

NFkB activation induced by MyD88



In vitro interaction between MyD88and Pd TLP TIR domains

GSTGST-mMyD88-

TIR

PdTLP-TIR

GSTGST-mMyD88-

TIR

hTLR2-TIR

GSTGST-hTLR2-

TIR

mMyD88-TIR

GSTGST-hTLR2-

TIR

PdTLP-TIR



future aims

• 3D structure of a prokaryote TIR protein

– Orientation of a TIR domain in the context of the full length prot.

• TIR virulence factors signaling mechanism

– Heterocomplex between Pd TIR and human MyD88-TIR domains

• Design compounds blocking TIR-TIR inter. – Ameliorate host chronic inflammation effects



Drugability of the TLR pathway:potential blocker of chronic inflammation signaling

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)



Acknowledgements

Ray Low

Siew Leong Chan

Robert C. Liddington

John C. Reed

Adam Godzik

Group Collaborators

project program grant update

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