Molecular Mimicry in Innate Immunity:

Structure of a Bacterial TIR

Virulence Factor

Jaime Pascual, Sanford-Burnham Institute

 Innate immunity:
first line of defense against microbes

Akira, Cell (2006)

Toll-like receptor signaling pathway


Suzuki, Trends in Immunology (2002)
Toll-like receptor activation mechanism
DeGrado, Structure (2008)
Crystal structure of monomeric human
Toll-like receptor 1 TIR domain

TIR 3D fold belongs

PDB_ID: 1FYV
to the flavodoxin-like
 family
that includes
bacterial proteins
Model for TIR-TIR signaling interaction:
homotypic via E-helix and heterotypic via BB loop

Jiang, PNAS (2006)

 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)

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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Bacteroidetes
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 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.
 Gel filtration chromatograms for PdTLP
aa1 Leu145 aa299
Helical bundle TIR
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
m AU

Helical bundle
-40

-50

-60

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

500
Monomer
400

300
C-terminal
m AU

200 TIR
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
PdTLP (FL) is a dimer, as observed by AUC

[0.5 mg/mL] [0.1 mg/mL] [0.05 mg/mL]

Dimerization constant: Kd = ~1.0 μM

1
H-15N 2D HSQC NMR spectrum of PdTLP
TIR-like domain
(ppm)

(ppm)
 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 Å
Space group: P 212121
Unit cell: a=80.8; b=85.6; c=89.6
PdTLP TIR structure has a TIR fold
Superposition of PdTLP-TIR domain onto human TLR1-TIR domain
(C RMSD = 2.8 Å)
 PdTLP TIR crystal dimer:
Exposed BB loops available for heterotypic interactions

Chain B
DD loop

BB loop
EE loop

Chain A

BB loop
 Dimerization Interface Determined Using
Deuterium Exchange Mass Spectrometry (DXMS)

PdTIR -
monomer

H H
B B full length
PdTLP -
dimer
 DXMS of PdTIR vs PdTLP:
Same protected interface in PdTLP as in the PdTIR crystal dimer
 DXMS of PdTIR vs PdTLP:
Same protected interface in PdTLP as in the PdTIR crystal dimer
Co-immunoprecipitation between
MyD88 and PdTLP TIR domains

ds
88

ac

a
yD

be
t
vc

Sm

ph
M
p

Se
Em
M.W.
4g 2g g 2g
(kDa)

*
37
IP-Myc
MyD88
IB-HA
26
*
*
19
IP-Myc
IB-Myc
PdTLP 15

37 *
MyD88 Lysate
IB-HA
26
Smac
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 increased
levels of pro-inflammatory cytokine secretion

Escherichia coli (Cirl, et al., 2008) :

TIR-like virulence factor directly interacts with human MyD88
 Drugability of the TLR pathway

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)

Model for virulence action by TIR mimicry
Healthy macrophage Infected macrophage
PAMP PAMP
LRR

TLR TLR

TIR
Death Domain

Helical bundle
MyD88
TIR virulence factor

kinase
IRAK
TIR

MyD88

NF-кB activation NF-кB activation

 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 PdTIR and human MyD88-TIR domains

 Acknowledgements
Group

Siew Leong Chan & Lieh Yoon Low

Collaborators

Eugenio Santelli (Robert C.

Liddington)

Gaelle Le Negrate (John C. Reed)

 Changes in H/D exchange upon dimerization (%D)

Deuteration level

-50% 0 50%

• Blue means the region become more protected upon dimerization; red means the region become less protected upon dimerization.
• No region is significantly less protected upon dimerization.
• Residues 259-286 are significantly more protected (change is bigger than 10%) upon dimerization.
•Pd-TIR crystal dimer interface observed between residues 260A-265A (DD loop & D helix) and 280B-288B (EE loop & E helix)
GST-pull down 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