Structural mimicry in virulent factors:

Bacteria TIR-like proteins

Jaime Pascual, Burnham Institute

 Innate immunity:
first line of defense against microbes

Akira, Cell (2006)

 Three arms for innate immune detection
of pathogens

Werts, Cell Death & Differentiation (2006)

Toll-like receptor signaling pathway

κ
Suzuki, Trends in Immunology (2002)
Toll-like receptor activation mechanism

Medzhitov, Cell (2007)

Crystal structure of monomeric human
Toll-like receptor 2 TIR domain

TIR 3D fold belongs

PDB_ID: 1FYV
to the flavodoxin-like
α/β family
that includes
bacterial proteins
Crystal structure of dimeric human
Toll-like receptor 10 TIR domain

Homodimer TIR-TIR
PDB_ID: 2J67 interactions mediated by
BB loops
Model for TIR-TIR signaling interaction:
receptor homodimer interacts with adaptors via BB loops

TIRAP
TLR10

TLR10
MyD88
PDB_ID: 2JS7

Gay, PLoS (2007)

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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of TIRs

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Phyla:
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R.palustris 5/340
RRPA0095 Y.pesti 7-308/348
G.me s y2426-2212
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N.p unct. p T.f t a llired 6310 13
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 Fusobacteria
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124-

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

• 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
H-15N 2D HSQC NMR spectrum of PdTLP
1

TIR-like domain
(ppm)

(ppm)
 Native protein crystal of
PdTLP TIR-like domain
Crystallization conditions at 4 C: Dataset collected in-house:
Salt: 0.2 M Ammonium sulfate Resolution: 2.75 A
Buffer: 0.1 M Sodium cacodylate pH 6.5 Space group: P 21212
Precipitant: 30% (w/v) PEG 8000 Unit cell: a=80.5; b=85.6; c=90.1
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
Effect of PdTLP protein on NFkB
activation induced by LPS
in MyD88 poc/poc MEFs

Luciferase reporter assay:

Cell lines (from B. Beutler @ Scripps) : MEFs WT and MyD88 poc/poc

Transfected constructs: luciferase reporter gene and
epitope tagged PdTLP
Cell stimulus: LPS
Chemi-luminescence read-out
Model for virulent action by TIR-like mimicry
healthy infected
cell cell
 Striking a balance through host mimicry:
Myxoma virus M11L virulent factor:
a Bcl-2 mimic binding and antagonizing pro-apoptotic Bax & Bak

Colman, Mol Cell (2007)

 Acknowledgements
Group Collaborators

Robert C. Liddington

Ray Low
John C. Reed

Siew Leong Chan

Adam Godzik