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Structural mimicry in virulent factors:

Bacteria TIR-like proteins

Jaime Pascual
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 M.acetivorans NP 617753.1-20091678 1-95/

M.barkeri ZP 00076529.1-23049344 1-95/18

distribution
M.mazei NP 632249.1-21226327 1-95/186
T.erythraeum Tery p 3677-23043038 111-21
T.elongatus tlr1460-22299003 335-441/860
N.punct. p 2340-23126020 6-112/523

of TIRs N.punct. p 802-23124438 2-104/511

B.abortus RBV02711
B.melitensis 114-217/250 114-217/2
BMEI1674-17987957
E.coli CFT073
S.enteritidis NP 754290.1-26248250
RSEN05207 102-206/228166-2
C.aurantiacus RCAU04526
S.aureus RSAY02557 2-91/91
138-242/280
C.tepidum CT1466-21674286 1-101/315
A.tumefaciens ymc-10955119
N.punct. p 3951-23127638 234-337/371
1-99/319
S.coeli. SC5G9.14-21218854 16-126/959N.punct. p 2323-23126003 2-102/308
S.coeli. SC7H1.23-21224291 4-112/913 S.sp. slr0658-16332137 2-107/331
S.coeli. StBAC16H6.31-21224333 30-137/93 R.solana. RSp0130-17548351 366-468/521
C.crescentus CC1413-16125662 3-124/688 M.loti msi144-2080397238-145/178
H.mobilis RHMO01074 2-117/813 B.suis BR0735-23501622 137-244/277
F.nucleatum ATCC 25586-19704057
Phyla:
B.melitensis BMEI1216-1798749938-145/178
G.metallireducens Gmet p 2197-23055336 9 C.thermocellum Chte p 1504-23021423 1-96
N.punct. p 809-23124445 283-387/412 B.fungorum Bcep p 4892-22986961 1-95/431
R.sp. y4lF-16519836 1-103/323 N.punct. NP 485998.1-17229450 232-329/10
X.axonopodis XAC3296-21244022 1-105/340 M.degradans Mdeg p 785-23026951 7-104/37
Euryarchaeota R.palustris RRPA00954 1-107/320
N.punct. p 232-23123829 1-106/247
Anabaena.sp RAN00789 1-102/363
N.punct. p 1378-23125040 1-97/371
N.punct. p 233-23123830 1-105/532 R.albus TIGR 207-308/348
N.punct. p 4631-23128321 1036-1144/1185
Proteobacteria N.punct. p 4808-23128502 960-1068/110
Y.pestis y2426-22126310 132-229/358
G.metallireducens Gmet p 2193-23055332 1
P.gingivalis RPG00640 1064-1166/1232 T.fusca Tfus p 2407-23019318 1-109/255
M.barkeri RMBA03625 195-295/969 H.sapiens SARM-14149667 523-629/690
N.punct. NP 485998.1-17229450 434-531/10 M.musculus
Firmicutes LOC195039-25056757 101-207/26
N.punct. NP 485998.1-17229450 1-99/1047 C.elegans 3E960-17553056 703-809/930
S.coeli. SC6A9.25-21223993 8-123/254 D.melanogaster CG7915-24660603 808-914/1
R.sp. y4cD-16519676 182-285/640 A.gambiae agCG54245-21298926 605-711/744
E.faecium REFA01101 73-174/283 S.coeli. SC6A9.38-21223984 1-99/886
Cyanobacteria H.pylori jhp1330-15612395
H.pylori
N.punct.HP1437-15646046
R.rubrum
p 4079-23127767
L.mesenteroides
74-161/187
Lmes
S.coeli. SCD82.03-21223014 1-99/966
81-181/239
S.coeli. RSX14993
25-133/425
S.coeli.
00014575.1-22966980
E.faecalis
1-95/191
SCC88.13c-21221060
REF01505
pRMN02248
189-23023477 1-103/274
124-
563-664/705
M.barkeri ZP
S.hominis B.subtilis
G.strain yddK-16077567
00078625.1-23051929
C.thermocellum
M.magneto.
S.mutans
RCTH02496
RMMA03087
BAB83481-18148891
RGEM10173 1-111/324
4-83/83 109-209/266
1-98/10
1-103/158
2-116/490
1-108/306
M.haemolytica RPQ02288 3-101/101
Chlorobi
Chloroflexi
Actinobacteria
Planctomycetes
 Fusobacteria
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

300
mAU Full length
200
(33 kDa)
100

0
0 20 40 60 80 100
ml

-10

-20 Oligomer
-30 N-terminal
Coiled-coil
-40
mAU
-50

-60

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

500
Monomer
400

300
C-terminal
mAU
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 rac occ us Bruc ella


In vitro interaction between MyD88
and PdTLP TIR domains
mMyD88- GST-hTLR2- GST
TIR 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
“Second life” avatar:
jamie pascal

See you in the metaverse…

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