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8/14/2019 project program grant update
http://slidepdf.com/reader/full/project-program-grant-update 1/25
Structural mimicry in virulence factors:
Bacteria TIR-like proteins
Jaime Pascual, Burnham Institute
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Toll-like receptor activation mechanismDeGrado, Structure (2008)
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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
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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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Sequence alignment between prokaryotic & eukaryotic TIRs
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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
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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
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Differential scanning calorimetry profiles forPd TLP full length, coiled-coil & TIR-like domains
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1H-15N 2D HSQC NMR spectrum of
Pd TLP TIR-like domain
( p p m )
(ppm)
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Pd TLP TIR-like domain is a TIRSuperposition of P. denitrificans Tlp-TIR domain onto human TLR1-TIR domain
(Cα RMSD = 2.8 Å)
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Pd TLP TIR-like crystal dimer:
Exposed BB loops available for heterotypic interactions
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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
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SalmonellaSalmonellaSalmonellaSalmonella ParacoccusParacoccusParacoccusParacoccus BrucellaBrucellaBrucellaBrucella
P. denitrificans TIR-like protein suppress
NFkB activation induced by MyD88
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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
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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
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Drugability of the TLR pathway:potential blocker of chronic inflammation signaling
Beutler, Nature (2004)
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BB loop mimic as inhibitor of prot-prot inter:a low molecular weight pyrrolidine interacts with MyD88 TIR
Rebek & Bartfai, PNAS (2006)
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Acknowledgements
Ray Low
Siew Leong Chan
Robert C. Liddington
John C. Reed
Adam Godzik
Group Collaborators
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