STP seminar 04/08
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Pd-TIR presentation by Leong

Pd-TIR presentation by Leong

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STP seminar 04/08 Presentation Transcript

  • 1. Structural studies of bacterial TIR- domain containing proteins: Paracoccus denitrificans Chan Siew Leong Pascual’s Lab Burnham Institute for Medical Research
  • 2. Innate immunityFirst line of defenseInnate immunity is the common mode of defenseagainst microorganisms, using limited set of pattern-recognition molecules.Rely on receptors to recognize microbial signaturessuch as LPS, CpG DNA, peptidoglycan and dsRNAfrom viruses.Toll-like receptors (TLR): Signal Transductionthrough the TIR (Toll/IL-1 receptor) domain
  • 3. Signaling of Toll-like ReceptorTrinchieri, 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) domainJin, M. S. et al. (2007). Cell 130: 1071Kim, H. M. et al. (2007). Cell 130: 906Brodsky, I. and R. Medzhitov (2007). Cell 130: 979
  • 5. Protein and non-protein ligands bind to different surfaces of TLR’s Leucine Rich-RegionJin, M. S. et al. (2007). Cell 130: 1071Kim, 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 TLR2Xu, 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 TRAMNunez 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 bacteriaCirl, 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 MyD88Cirl, 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)MSANDRAIETLRREIAKLQTDGAAIARKDAGIRAKLASAMAAQAKAKTAPALRLKQAEASRLEKELMATSKSQADIATKIAKKQSSLSAKLVVQANEAKKADAKAKKNQERVSKTQEEATRKLEAGYRKLTLENQSLEQRLQRELSAMKPTAGPTTNADLTSAPPHDIFISHAWEDKADFVEALAHTLRAAGAEVWYDDFSLRPGDSLRRSIDKGLGSSRFGIVVLSTHFFKKEWPQKELDGLFQLESSGRSRILPIWHKVSKDEVASFSPTMADKLAFNTSTKSVDEIVADLMAIIRD16.9kDa; 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 domainsLow, 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
  • 20. PdTLP!s TIR domain binds to TLR4 and MyD88 TIR domainsLow, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481
  • 21. Microbial Pathogens Utilize Structural Mimicry To Manipulate Host Cellular Functions
  • 22. Plant TIR Domains • p50 effector associates with TIR domain (LRR?) • Mechanism of signaling is not knownBurch-Smith, T. M. and S. P. Dinesh-Kumar (2007). Science 401: pe46
  • 23. 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
  • 24. 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
  • 25. Future workDetermine crystal structure of TIR-Like Protein fromParacoccus denitrificansStructures of TLP from other organisms (E. coli, Brucella,Yersinia, Agrobacterium, Arabidopsis)Do bacterial TIR domains use BB-loop to bind to MyD88and other adaptors?Interactions between TIR domains and in-complex withother adaptors
  • 26. Acknowledgement... Jaime Pascual Ray Low and Eugenio Santelli Sarah Hamilton Yvonne Thank You