Structural studies of bacterial TIR-   domain containing proteins:     Paracoccus denitrificans                Chan Siew Le...
Innate immunityFirst line of defenseInnate immunity is the common mode of defenseagainst microorganisms, using limited set...
Signaling of Toll-like ReceptorTrinchieri, G. and A. Sher (2007). Nat. Rev. Immunol. 7: 179
Models of Ligand-Induced TLR Activation                                   Leucine-Rich Repeats (LRR)                Toll/I...
Protein and non-protein ligands bind to different surfaces of                TLR’s Leucine Rich-RegionJin, M. S. et al. (2...
Human TIR-domain-containing Adaptor Family
Crystal structures of TIR-domains of TLR1 and TLR2                                       N   C                        BB  ...
TLR10 TIR domains homodimerize via BB-Loop                   BB                                    PDB: 2j67              ...
A Model for TIR-TIR domains signaling: TLR4 homodimer                      docked with MAL and TRAM                       ...
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 intracellula...
TIR-Containing Proteins (E. coli and B. melitensis) impair TLR                   signaling and interact with MyD88Cirl, C....
Objectives: Determine 3D Structure of prokaryote TIR domain Prokaryote TIR vs. Eukaryote TIR Interaction between Prokaryot...
TIR-Like Proteins from Paracoccus denitrificans (PdTLP)>PdTLP(Paracoccus denitrificans)MSANDRAIETLRREIAKLQTDGAAIARKDAGIRAKL...
PdTLP is composed of two independent folded domainsLow, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481
PdTIR showed well-dispersed 2D 15N-HSQC spectra
Crystallization PdTIR                                       PdTIR on Native Gel• Protein concentration 5.3 mg/ml in buffer...
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 8...
PdTLP!s TIR domain binds to TLR4 and MyD88 TIR domainsLow, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481
Microbial Pathogens Utilize Structural Mimicry To      Manipulate Host Cellular Functions
Plant TIR Domains                                                    • p50 effector associates with TIR                   ...
Arabidopsis TIR-Containing Protein exists as monomer and dimer  Dynamic Light Scattering (DLS)            Native Gel      ...
Arabidopsis TIR-Containing Protein exists as monomer under                    reducing conditions Dynamic Light Scattering...
Future workDetermine crystal structure of TIR-Like Protein fromParacoccus denitrificansStructures of TLP from other organis...
Acknowledgement... Jaime Pascual Ray Low and Eugenio Santelli Sarah Hamilton Yvonne             Thank You
STP seminar 04/08
STP seminar 04/08
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STP seminar 04/08

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STP seminar 04/08

  1. 1. Structural studies of bacterial TIR- domain containing proteins: Paracoccus denitrificans Chan Siew Leong Pascual’s Lab Burnham Institute for Medical Research
  2. 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. 3. Signaling of Toll-like ReceptorTrinchieri, G. and A. Sher (2007). Nat. Rev. Immunol. 7: 179
  4. 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. 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. 6. Human TIR-domain-containing Adaptor Family
  7. 7. Crystal structures of TIR-domains of TLR1 and TLR2 N C BB Loop TLR1 TLR2Xu, Y. et al. (2000) Nature 408: 111
  8. 8. TLR10 TIR domains homodimerize via BB-Loop BB PDB: 2j67 Loop
  9. 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. 10. TIR-domains are also present in prokaryotes
  11. 11. • What role does TIR domain play in bacteria?• An evolved mechanism to interfere with host’s innate immune responses?
  12. 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. 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. 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. 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. 16. PdTLP is composed of two independent folded domainsLow, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481
  17. 17. PdTIR showed well-dispersed 2D 15N-HSQC spectra
  18. 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. 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. 20. PdTLP!s TIR domain binds to TLR4 and MyD88 TIR domainsLow, L. Y. et al. (2007). Biochem. Biophy. Res. Comm. 356: 481
  21. 21. Microbial Pathogens Utilize Structural Mimicry To Manipulate Host Cellular Functions
  22. 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. 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. 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. 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. 26. Acknowledgement... Jaime Pascual Ray Low and Eugenio Santelli Sarah Hamilton Yvonne Thank You
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