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Structural mimicry in virulent factors:

Bacteria TIR-like proteins

Jaime Pascual, Burnham Institute


Innate immunity:
first line of defense against microbes
Three arms for innate immune detection
of pathogens
Toll-like receptor signaling pathway

κ
Suzuki et al, Trends in Immunology, 2002
Crystal structure of human Toll-like
receptor 2 TIR domain

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
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
Phylogenetic

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 Fusobacteria
3477

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124-

Bacteroidetes
Metazoa
aims
• 3D structure of a prokaryote TIR domain
– Comparison with a eukaryote TIR receptor

• TIR signaling: prokar – eukar complex


– Homo- vs. hetero-typic interactions

• Function of TIR-like domain in bacteria


– Prevent host cell inflammation signals (virulent factor)

– Prokaryote programmed cell death


Prokaryote TIR-like proteins
expression trials in E. coli
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, toxic
Staphylococus aureus Cloned, no expression

P. denitrificans TIR-like protein (PdTLP)


could only be expressed in high quantity
using the Rosetta(DE3)-pLysS E. coli strain.
Limited proteolysis analysis of PdTLP
Mass spectrometry identification of
PdTLP digestion fragments

Full length

C-terminal
fragment
(S146-A-M-K-P)
Gel filtration chromatograms for PdTLP
aa1 Leu145 aa299
Coiled­coil TIR­like
pI 10.2 Full length pI 9.6 pI 5.7
600

158kDa 44kDa 17kDa


500
Dimer
400

Full length
mAU

300

200
(33 kDa)
100

0
0 20 40 60 80 100
ml

-10

-20 Oligomer
-30 N-terminal
mAU

Coiled-coil
-40

-50

-60

-70
(16 kDa)
-80
0 20 40 60 80 100
ml
600

500
Monomer
400
C-terminal
mAU

300

200 TIR-like
100
(17 kDa)
0

-100
0 20 40 60 80 100
ml
Differential scanning calorimetry profiles for
PdTLP full length, coiled-coil & TIR-like domains
Circular dichroism spectrum of
the coiled-coil region of PdTLP
H-15N 2D HSQC NMR spectrum of PdTLP
1

TIR-like domain
(ppm)

(ppm)
Prokaryote TIR-like proteins suppress
NFkB activation induced by MyD88

Salmo ne lla Pa raco cc us Bruc el la


In vitro interaction between MyD88
and PdTLP TIR domains
mMyD88-TIR GST-hTLR2- GST
TIR

hTLR2-TIR GST-mMyD88- GST


TIR

PdTLP-TIR GST-hTLR2- GST


TIR

PdTLP-TIR GST-mMyD88- GST


TIR
Model for virulent action by TIR-like mimicry
healthy infected
cell cell
Striking a balance through host mimicry:
Salmonella SopE (GEF) & SptP (GAP) virulent factors

Stebbins, C. Nature 412:701-5 (2001)


Drugability of the TLR pathway

Beutler, B. Nature 430:257-263 (2004)


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

Davis, CN, Rebek, J & Bartfai, B. PNAS 103:2953-2958 (2006)


Acknowledgements
Group Collaborators

Ray Low
Robert C. Liddington
Genaro Pimienta
John C. Reed
Takashi Mukasa
Adam Godzik

Funding: R21AI065602 (NIAID-NIH )


“Second life” avatar:
jamie pascal

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