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
distribution
of TIRs
Phyla:
Euryarchaeota
Proteobacteria
Firmicutes
Cyanobacteria
Chlorobi
Chloroflexi
Actinobacteria
Planctomycetes
 Fusobacteria
Bacteroidetes
Metazoa

M.acetivorans NP 617753.1-20091678 1-95/
M.barkeri ZP 00076529.1-23049344 1-95/18
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

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

B.abortus
RBV02711
114-217/250 114-217/2
B.melitensis
BMEI1674-17987957
E.coli
CFT073
NP
754290.1-26248250
166-2
S.enteritidis
RSEN05207
102-206/228
C.aurantiacus
RCAU04526
2-91/91
S.aureus RSAY02557
138-242/280
C.tepidum CT1466-21674286 1-101/315
A.tumefaciens
ymc-10955119
234-337/371
N.punct. p 3951-23127638
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
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
R.palustris RRPA00954 1-107/320
Anabaena.sp RAN00789 1-102/363
N.punct. p 232-23123829 1-106/247
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
Y.pestis
y2426-22126310 132-229/358
N.punct. p 4808-23128502 960-1068/110
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
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
H.pylori
jhp1330-15612395
74-161/187
S.coeli. SCD82.03-21223014 1-99/966
H.pylori
HP1437-15646046
81-181/239
S.coeli.
RSX14993
1-95/191
N.punct.
p 4079-23127767
25-133/425
S.coeli.
SCC88.13c-21221060
563-664/705
R.rubrum
00014575.1-22966980
E.faecalis
REF01505
1-103/274
L.mesenteroides
Lmes
pRMN02248
189-23023477
124B.subtilis
yddK-16077567
109-209/266
M.barkeri
ZP
00078625.1-23051929
1-98/10
C.thermocellum
M.magneto.
S.mutans
RCTH02496
RMMA03087
1-111/324
4-83/83
1-103/158
S.hominis
G.strain
BAB83481-18148891
RGEM10173
2-116/490
1-108/306
M.haemolytica
RPQ02288
3-101/101

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
Bacillus subtilis
Brucella abortus
Burkholderia fungorum
Escherichia coli CFT073
Paracoccus denitrificans
Salmonella enteriditis
Staphylococus aureus

Status
Cloned
Cloned, no expression
Cloned, no expression
Cloned, toxic, refolded, unstable
Cloned, expressed, purified, >20mg/ml, xtal trials
Cloned, toxic
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

Coiled­coil
pI 10.2

600

Dimer

500

TIR­like

Full length pI 9.6
158kDa

44kDa

pI 5.7

17kDa

400

Full length
(33 kDa)

300
mAU
200
100
0
0

20

40

60

80

100

ml
0
-10

Oligomer

-20

N-terminal
Coiled-coil
(16 kDa)

-30
-40
mAU
-50
-60
-70
-80
0

20

40

60

80

100

ml

600

Monomer

500

C-terminal
TIR-like
(17 kDa)

400
300
mAU
200
100
0
-100
0

20

40

60
ml

80

100

aa299

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
TIR-like domain

(ppm)

1

(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
mMyD88TIR

GST-hTLR2TIR

hTLR2-TIR

GST-mMyD88TIR

PdTLP-TIR

GST-hTLR2TIR

PdTLP-TIR

GST-mMyD88TIR

GST

GST

GST

GST

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

Ray Low
Genaro Pimienta
Takashi Mukasa

Collaborators

Robert C. Liddington
John C. Reed
Adam Godzik

“Second life” avatar:
jamie pascal
See you in the metaverse…

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