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

PDB_ID: 1FYV

TIR 3D fold belongs to the flavodoxin-like α/β family that includes bacterial proteins

Crystal structure of dimeric human Toll-like receptor 10 TIR domain

PDB_ID: 2J67

Homodimer TIR-TIR interactions mediated by BB loops

Model for TIR-TIR signaling interaction:
receptor homodimer interacts with adaptors via BB loops
TIRAP

TLR10

MyD88 PDB_ID: 2JS7

TLR10

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

B.abortus RBV02711 114-217/250 B.melitensis BMEI1674-179 E.coli C 87957 114-21 FT073 7/2 S.en NP 754 290.1-2 C.a teritidis 624825 ur a RSE 0 166-2 S.a nt ia N052 cus 07 10 C. ure RCA 2-206 A. tepid us RS /228 U0 4 AY u N tu 526 025 2-91 N .p me m C 57 / 91 S. .pu unc faci T14 138 66 en sp n t. -24 2/2 . s ct. p 3 s ym 2167 80 95 p 42 lr0 2 c-1 186 09 65 32 23 1- 1 55 8- 3-2 12 01 11 /31 76 16 3 92 5 38 33 1 26 34 1-33 21 00 99 7/3 3 37 /3 271 19 210 10 2/ 30 7/ 33 8 1

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

7 04 /1 10 99 1/ 153 0 445 43 29 0 45 72 9 -1 29 /96 .1 32 72 95 -1 98 /12 -2 66 95 59 8.1 -11 10 51 48 99 64 8/1 62 P 485 10 106 N 03 85 40 60t. NP BA /11 06 29 nc 144 t. RM PG0 850 6-1 pu nc ri R 312 103 N. .pu rke alis 8-2 321 2 N ba iv p 480 128 5/53 -23 M. ing 1-10 ct. 631 P.g 3830 un p4 47 2312 N.p unct. 2331-106/2 N.p unct. p 23829 32-231 N.p ct. p 2 4 1-107/320 N.pun RRPA0095 R.palustris 5/340 X.axonopodis XAC3296-21244022 1-10 R.sp. y4lF-16519836 1-103/323 N.punct. p 809-23 G.m 124445 2 e 83-387/4 F.n tallireduc 12 ens G H. uclea me t p 21 C. mobi tum A 97-23 S cre lis RH TCC 25 05533 5 MO 69 86-1 S .co sce 010 970 S. .co eli. ntus 4057 74 c 2-1 oe eli. StBA CC1 17/ 41 li. SC C 813 316 SC 7H 16H 12 6.3 5G 1.2 56 1-2 3 62 9 3-1 12 .1 -212 24 424 24 /68 33 21 29 3 8 2 1 30 18 4 -13 7/9 3

85 4

1

612 6

-1 1

/9 5

2/9

9

13

Phylogenetic distribution of TIRs

5/ 1-9 95/18 6 678 091 344 1 5/18 23 -20 49 1-9 /5 0 53.1 230 27 112 /86 177 .163 6- 41 P6 529 22 20 sN -4 ran 076 -21 60 35 1 i vo 00 9.1 312 3 3 1-2 c et ZP 24 M.a -2 00 11 e ri 32 40 ark 8 99 P6 23 22 303 M.b iN p -2 ze t. 4 60 ma nc 30 M. 14 pu -2 tlr N. 77 s tu 36 p ga ry on el Te T. m
.p u nc t. p 80 223 12 44 38 210 4/ 51 1

e T. ry

eu ra th
N

78 3 /1 77 48 45 78 5 1 4/2 5/1 817 24 -14 23 077 3 38 13 6 99 01 039 1-9 22 74 23 16 Sp 208 98 R 4214 50 17 0 3 a. 4 16 -23 -2 /431 I12 an si1 35 504 1-95 ol i m R07 BME te p 1 961 6 s 298 R. .lot is B nsis m Ch /10 92-2 -329 M .su lite ellu p 48 0 232 B .me moc cep 2945 172 7 B ther rum B 98.17-104/3 C. ungo 4859 026951 B.f unct. NP 785-23 deg p N.p dans M 102/363 M.degra RAN00789 1p Anabaena.s N.punct. p 1378-23125040 1-97/371 R.albus TIGR 20 7-308/348 Y.pesti G.me s y2426-2212 6310 13 tallir T.f educ 2-229/3 58 ens G H.s usca T fus met p api M. p 24 ens 2193 C. mus 07-2 -2305 301 D ele cu SARM 5332 931 1 A .m ga lu -14 8 1149 S. .ga ela ns s LO 109 C1 667 no 3E co m /255 95 96 523 el bi a ga 03 0-1 -62 s i. 9-2 9/6 7 SC e a ter 50 90 gC CG 553 56 05 6A 75 G 79 67 71 9. 54 15 01 03 38 24 -20 -2 -80 46 -2 57/2 9/9 21 12 60 6 30 60 29 23 38 89 98 08 26 4 -9 160 14 99 5/1 /8 71 86 1/ 74 4

51

36

6-

8 46

2 /5

1

66 /9 99 105 4 4/7 01 66 23 391 12 56 /1 -2 60 95 03 10 12. 4 3 22 /27 66 D8 499 c-21 03 9/2 SC X1 .13 -20 1-1 S 8 i. 109 05 el . R C8 15 567 co i F0 el SC 077 S. .co eli. s RE 83 -16 -83/ S .co cali dK 58 48 4 S ae lis yd 03/1 022 7 1-1 E.f ubti s RMN 0308 B.s utan MMA 16/490 to. R S.m 173 2 -1 agne M.m in RGEM10 8 3-101/101 G.stra ica RPQ0228 M.haemolyt S.hominis BAB83481-18148891 1-108/306 C.thermocellum RC TH02496 1-111/32 M.barke 4 ri ZP 0007862 L.m e 5.1-2305 R.ru senteroid 1929 1-9 es Lm 8/10 N.p brum es p 0 001 189-2 H. unct 457 3023 5 H py lo . p 4 .1-2 477 1 296 24E .py ri HP 079-2 698 3 12 R .fa lor 14 0 776 3 S .s ec i jh 7 .c p. ium p13 7-15 2564 oe y4 30 133 60 -1 / 42 li. cD RE 46 5 81 SC -1 FA0 5612 -18 39 11 6 A 65 1 1/2 57 01 9 39 9 4-1 73 .2 676 61 5 17 18 -2 /18 4/ 1 27 28 3 /6 40
22 39
28 5

93

812 3

/2 5

4

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 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

Coiled­coil
pI 10.2
600 500 400 300 200 100 0 0 20 40 ml 0 -10 -20 -30 mAU -40 -50 -60 -70 -80 0 600 500 400 mAU 300 200 100 0 -100 0 20 40 ml 20 40 ml

Leu145 Full length pI 9.6 158kDa 44kDa 17kDa

TIR­like
pI 5.7

aa299

Dimer

Full length (33 kDa)
60 80 100

mAU

Oligomer

N-terminal Coiled-coil (16 kDa)
60 80 100

Monomer

C-terminal TIR-like (17 kDa)
60 80 100

Differential scanning calorimetry profiles for
PdTLP full length, coiled-coil & TIR-like domains

1

H-15N 2D HSQC NMR spectrum of PdTLP TIR-like domain

(ppm)

(ppm)

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

In vitro interaction between MyD88 and PdTLP TIR domains
mMyD88-TIR GST-hTLR2TIR GST

hTLR2-TIR

GST-mMyD88TIR

GST

PdTLP-TIR

GST-hTLR2TIR

GST

PdTLP-TIR

GST-mMyD88TIR

GST

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

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