Pd-TIR presentation by Leong

1. Structural studies of bacterial TIR-
domain containing proteins:
Paracoccus denitrificans
Chan Siew Leong
Pascual’s Lab
Burnham Institute for Medical Research

2. 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

3. Signaling of Toll-like Receptor
Trinchieri, G. and A. Sher (2007). Nat. Rev. Immunol. 7: 179

4. Models of Ligand-Induced TLR Activation
Leucine-Rich Repeats (LRR)
Toll/IL-1 Receptor (TIR) domain
Jin, M. S. et al. (2007). Cell 130: 1071
Kim, H. M. et al. (2007). Cell 130: 906
Brodsky, I. and R. Medzhitov (2007). Cell 130: 979

5. Protein and non-protein ligands bind to different surfaces of
TLR’s Leucine Rich-Region
Jin, M. S. et al. (2007). Cell 130: 1071
Kim, H. M. et al. (2007). Cell 130: 906

6. Human TIR-domain-containing Adaptor Family

7. Crystal structures of TIR-domains of TLR1 and TLR2
N C
BB
Loop
TLR1
TLR2
Xu, Y. et al. (2000) Nature 408: 111

8. TLR10 TIR domains homodimerize via BB-Loop
BB
PDB: 2j67
Loop

9. A Model for TIR-TIR domains signaling: TLR4 homodimer
docked with MAL and TRAM
MAL
TLR4
TLR4
TRAM
Nunez Miguel R. et al. (2007) PLoS One 2(8):e788

10. TIR-domains are also present in prokaryotes

11. • What role does TIR domain play in bacteria?
• An evolved mechanism to interfere with host’s innate
immune responses?

12. 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

13. 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

14. Objectives:
Determine 3D Structure of prokaryote TIR domain
Prokaryote TIR vs. Eukaryote TIR
Interaction between Prokaryote TIR and Eukaryote TIR
Plant TIR domains

15. TIR-Like Proteins from Paracoccus denitrificans (PdTLP)
>PdTLP(Paracoccus denitrificans)
MSANDRAIETLRREIAKLQTDGAAIARKDAGIRAKLASAMAAQAKAKTAPALRLKQAEASRLEKELMATSKSQADIATKIAKKQSSLSAKLVVQ
ANEAKKADAKAKKNQERVSKTQEEATRKLEAGYRKLTLENQSLEQRLQRELSAMKPTAGPTTNADLTSAPPHDIFISHAWEDKADFVEALAHTL
RAAGAEVWYDDFSLRPGDSLRRSIDKGLGSSRFGIVVLSTHFFKKEWPQKELDGLFQLESSGRSRILPIWHKVSKDEVASFSPTMADKLAFNTS
TKSVDEIVADLMAIIRD
16.9kDa; 154 a.a; 3M; ! = 23490
1 2 3 4 5 6
Lane 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
Chymotrypsin cleavage
Coiled-coil domain TIR

16. PdTLP is composed of two independent folded domains
Low, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481

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

18. Crystallization PdTIR
PdTIR on Native Gel
• 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

19. 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

21. PdTLP!s TIR domain binds to TLR4 and MyD88 TIR domains
Low, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481

22. Microbial Pathogens Utilize Structural Mimicry To
Manipulate Host Cellular Functions

24. Plant TIR Domains
• p50 effector associates with TIR
domain (LRR?)
• Mechanism of signaling is not
known
Burch-Smith, T. M. and S. P. Dinesh-Kumar (2007). Science 401: pe46

25. Arabidopsis TIR-Containing Protein exists as monomer and dimer
Dynamic Light Scattering (DLS) Native Gel
8.6 5.0 2.5 mg/ml
•Two species, non homogenous
•Poor Polyindex ~ 0.6

26. Arabidopsis TIR-Containing Protein exists as monomer under
reducing conditions
Dynamic Light Scattering (DLS) Native Gel
1.0 5.0 7.5 10.6
mg/ml
•Homogenous
•PolyIndex = 0.26

27. 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

28. Acknowledgement...
Jaime Pascual
Ray Low and Eugenio Santelli
Sarah Hamilton
Yvonne
Thank You