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