Structural studies of bacterial TIR-

domain containing proteins:

Paracoccus denitrificans

Chan Siew Leong

Pascual’s Lab

Burnham Institute for Medical Research

Innate immunity

First line of defense

Innate immunity is the common mode of defense

against microorganisms, using limited set of pattern-

recognition molecules.

Rely on receptors to recognize microbial signatures

such as LPS, CpG DNA, peptidoglycan and dsRNA

from viruses.

Toll-like receptors (TLR): Signal Transduction

through the TIR (Toll/IL-1 receptor) domain

Trinchieri, G. and A. Sher (2007). Nat. Rev. Immunol. 7: 179

Signaling of Toll-like Receptor

Models of Ligand-Induced TLR Activation

Brodsky, I. and R. Medzhitov (2007). Cell 130: 979

Toll/IL-1 Receptor (TIR) domain

Leucine-Rich Repeats (LRR)

Kim, H. M. et al. (2007). Cell 130: 906

Jin, M. S. et al. (2007). Cell 130: 1071

Kim, H. M. et al. (2007). Cell 130: 906

Jin, M. S. et al. (2007). Cell 130: 1071

Protein and non-protein ligands bind to different surfaces of

TLR’s Leucine Rich-Region

Human TIR-domain-containing Adaptor Family

TLR1

TLR2

Xu, Y. et al. (2000) Nature 408: 111

BB

Loop

N C

Crystal structures of TIR-domains of TLR1 and TLR2

TLR10 TIR domains homodimerize via BB-Loop

PDB: 2j67BB

Loop

TRAM

MAL

TLR4

TLR4

A Model for TIR-TIR domains signaling: TLR4 homodimer

docked with MAL and TRAM

Nunez Miguel R. et al. (2007) PLoS One 2(8):e788

TIR-domains are also present in prokaryotes

• What role does TIR domain play in bacteria?

• An evolved mechanism to interfere with host’s innate

immune responses?

TIR-Containing Proteins (E. coli and B. melitensis) reduce cytokine

secretion and increases accumulation of intracellular bacteria

Cirl, C. et al. (2008). Nat. Med. published online 9 March 2008; doi:10.1038/nm1734

TIR-Containing Proteins (E. coli and B. melitensis) impair TLR

signaling and interact with MyD88

Cirl, C. et al. (2008). Nat. Med. published online 9 March 2008; doi:10.1038/nm1734

Objectives:

Determine 3D Structure of prokaryote TIR domain

Prokaryote TIR vs. Eukaryote TIR

Interaction between Prokaryote TIR and Eukaryote TIR

Plant TIR domains

1 2 3 4 5 6Lane 1: MW Marker

Lane 2: Cell Lysate

Lane 3: After IPTG induction

Lane 4: Full Length PdTLP (after His-

Trap and S200 Gel Filtration)

Lane 5: After Chymotripsin Digestion

Lane 6: After final S-200 Gel

Filtration

Coiled-coil domain TIR

Chymotrypsin cleavage

>PdTLP(Paracoccus denitrificans)

MSANDRAIETLRREIAKLQTDGAAIARKDAGIRAKLASAMAAQAKAKTAPALRLKQAEASRLEKELMATSKSQADIATKIAKKQSSLSAKLVVQ

ANEAKKADAKAKKNQERVSKTQEEATRKLEAGYRKLTLENQSLEQRLQRELSAMKPTAGPTTNADLTSAPPHDIFISHAWEDKADFVEALAHTL

RAAGAEVWYDDFSLRPGDSLRRSIDKGLGSSRFGIVVLSTHFFKKEWPQKELDGLFQLESSGRSRILPIWHKVSKDEVASFSPTMADKLAFNTS

TKSVDEIVADLMAIIRD

16.9kDa; 154 a.a; 3M; ! = 23490

TIR-Like Proteins from Paracoccus denitrificans (PdTLP)

Low, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481

PdTLP is composed of two independent folded domains

PdTIR showed well-dispersed 2D 15N-HSQC spectra

Crystallization PdTIR

• Protein concentration 5.3 mg/ml in buffer 10 mM Tris pH 8.0

• Room temperature and 4°C

• Screening: Classics, PEGS, Hampton I & II, Wizard I & II,

MPD, PACT, Cryos, JCSG+ Screening Suites.

• 0.1 M Sodium cacodylate pH 6.5, 0.2 M Ammonium sulfate

and 30% PEG 8000

PdTIR on Native Gel

X-ray diffraction of Pd TIR-domain crystals: 2.4 Å

• 0.1 M Sodium cacodylate pH 6.5, 0.2 M Ammonium sulfate and 30% PEG 8000

• Sitting drop, 4°C, 7 days

• Orthorhombic crystal system; P212121 space group; a=80.78, b=85.61; c=89.62

Low, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481

PdTLP!s TIR domain binds to TLR4 and MyD88 TIR domains

Microbial Pathogens Utilize Structural Mimicry To Manipulate Host Cellular Functions

Burch-Smith, T. M. and S. P. Dinesh-Kumar (2007). Science 401: pe46

Plant TIR Domains

• p50 effector associates with TIR

domain (LRR?)

• Mechanism of signaling is not

known

Arabidopsis TIR-Containing Protein exists as monomer and dimer

Dynamic Light Scattering (DLS)

•Two species, non homogenous

•Poor Polyindex ~ 0.6

Native Gel

8.6 5.0 2.5 mg/ml

Arabidopsis TIR-Containing Protein exists as monomer under

reducing conditions

Dynamic Light Scattering (DLS)

•Homogenous

•PolyIndex = 0.26

Native Gel

1.0 5.0 10.6

mg/ml7.5

Future work

Determine crystal structure of TIR-Like Protein from

Paracoccus denitrificans

Structures of TLP from other organisms (E. coli, Brucella,

Yersinia, Agrobacterium, Arabidopsis)

Do bacterial TIR domains use BB-loop to bind to MyD88

and other adaptors?

Interactions between TIR domains and in-complex with

other adaptors

Acknowledgement...

Jaime Pascual

Ray Low and Eugenio Santelli

Sarah Hamilton

Yvonne

Thank You