TIR faculty seminar 090909

8/14/2019 TIR faculty seminar 090909

1/20

Molecular Mimicry in Innate Immunity:

Structure of a Bacterial TIRVirulence Factor

Jaime Pascual, Burnham Institute


2/20

Suzuki, Trends in Immunology (2002)

Toll-like receptor signaling pathway


3/20

Toll-like receptor activation mechanismDeGrado, Structure (2008)


4/20

TIR 3D fold belongsto the flavodoxin-like

/ familythat includes

bacterial proteins

Crystal structure of monomeric humanToll-like receptor 1 TIR domain

PDB_ID: 1FYV


5/20

Model for TIR-TIR signaling interaction:homotypic via E-helix and heterotypic via BB loop

Jiang, PNAS (2006)


6/20

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)


7/20

Sequence alignment between prokaryotic & eukaryotic TIRs


8/20

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

Space group: P 2 12121

Unit cell: a=80.8; b=85.6; c=89.6


9/20

Pd TLP TIR structure has a TIR foldSuperposition of

Pd

TLP-TIRdomain onto human TLR1-TIRdomain (C RMSD = 2.8 )


10/20

Chain A

Chain B

BB loop

BB loop

DD loop

EE loop

Pd TLP TIR crystal dimer :Exposed BB loops available for hetero typic interactions


11/20

0

100

200

300

400

500

600

0 20 40 60 80 100

m A U

ml

-80

-70

-60

-50

-40

-30

-20

-10

0

0 20 40 60 80 100

m A U

ml

-100

0

100

200

300

400

500

600

0 20 40 60 80 100

m A U

ml

158kDa 44kDa 17kDa

Gel filtrationchromatograms for Pd TLP

Full length(33 kDa)

C-terminalTIR(17 kDa)

N-terminalHelical bundle(16 kDa)

Dimer

Monomer

Oligomer

aa299

TIRHelical bundlepI 5.7pI 10.2 Full length pI 9.6

Leu145aa1 aa299


Leu145aa1 aa299


Leu145aa1


12/20

Dimerization Interface Determined UsingDeuterium Exchange Mass Spectrometry ( DXMS)

Pd TIR - monomer

full lengthPd TLP -

dimer

HB

HB


13/20

DXMS of Pd TIR vs Pd TLP:Same protected interface in Pd TLP as in the Pd TIR crystal dimer


14/20

DXMS of Pd TIR vs Pd TLP:Same protected interface in Pd TLP as in the Pd TIR crystal dimer


15/20


16/20

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 increasedlevels of pro-inflammatory cytokine secretion

Escherichia coli (Cirl, et al., 2008) :TIR-like virulence factor directly interacts with human MyD88


17/20

Drugabilityof the TLR pathway

Beutler, Nature (2004)


18/20

BB loop mimic as inhibitor of prot-prot inter:a low molecular weight pyrrolidine interacts with MyD88TIR

Rebek & Bartfai, PNAS (2006)


19/20

L R R

D e a t

h

D o m a i n

MyD88

NF-B activation NF-B activation

PAMP

TLR

TIR

Healthy macrophage Infected macrophage

TIR virulence factor

H e

l i c a

l

b u n

d l

e

TIR

Model for virulence action by TIR mimicry

IRAKkinase

MyD88

TLR

PAMP


20/20

Acknowle dgements

Siew Leong Chan & Lieh Yoon Low

Eugenio Santelli (Robert C.Liddington)

Gaelle Le Negrate (John C. Reed)

Group

Collaborators

Documents

TIR faculty seminar 090909