Comparative Modeling and Functional Analysis of Toll-Like Receptor Ligand-Recognition Domains
- 주제(키워드) Computational biology
- 발행기관 아주대학교
- 지도교수 Sangdun Choi
- 발행년도 2012
- 학위수여년월 2012. 2
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000012393
- 본문언어 한국어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Toll-like receptors (TLRs) play a central role in bridging the innate and adaptive immune responses. Despite the wide range of ligands recognized by TLRs, the receptor shares a common structural framework in their extracellular, ligand-recognition domains. The domains adopt horseshoe-shaped structures that are built from leucine-rich repeats. TLRs signal via common pathways that lead to the expression of diverse inflammatory genes. In addition, each TLR elicits specific cellular responses to pathogens owing to the differential usage of intracellular adapter proteins. TLR signaling pathways are also implicated in serious autoimmune diseases such as endotoxic shock and thus are important therapeutic targets. To date, only five crystallographic structures of TLRs Ectodomain (ECD) interactions with cognate ligands have been reported using X-ray diffraction or nuclear magnetic resonance (NMR) spectroscopy experiments. These crystal structures have revealed the shape of the receptors and how they bind to their cognate ligands. Indeed, identification of all TLR-ligand receptor interaction of each TLR member using crystallographic and NMR studies have proven to be very difficult. Owing to the lack of TLR structural information, explanations for ligand-binding mechanisms have been difficult to provide. Computational modeling enables initial predictions of three-dimensional structures for the investigation of receptor-ligand interaction mechanisms. In order to understand the nature of the interactions of the TLR ECD with their ligands, we constructed three dimensional structures of human TLR10, human TLR8, mouse TLR8, rat TLR8, bovine TLR8 and porcine TLR8 and refined the model through molecular dynamics (MD) simulations. Subsequently, models of essential complexes involved in the TLR signaling processes were yielded through protein-protein docking analysis. The docked complexes of the heterodimer of human TLR10/1, human TLR10/2 and homodimer of human TLR10 were then used for identifying the interaction with lipopeptide ligands such as Pam3CSK4 and PamCysPamSK4 using MOE-Dock and ACE-dock. The multiple protein-ligand docking studies revealed that Pam3CSK4 might be the possible ligand for the human TLR10/2 and PamCysPamSK4, a di-acylated lipopeptide, might activate the human TLR10/1 and homodimer of human TLR10. Computational modeled structures of TLR10/2, TLR10/1, and homodimer of TLR10 complexes and mutagenesis studies reveal similarities, as well as some subtle differences, in the ligand binding and dimer interface in comparison with the available crystal structure of TLR2/1-lipopeptides. The primary sequence of rodent and non-rodent TLR8s are similar, but the antiviral compound (R848) that activates the pathway is species-specific and the factors that underlying this receptor？s species-specificity remains unknown. In addition, the molecular docking complexes of the homodimer of human TLR8, mouse TLR8, rat TLR8, bovine TLR8 and porcine TLR8 were used for interaction with anti-viral compound. These comparative analyses of modeled TLR8 and docking studies observation suggests the hypothesis that the undefined region that is in close proximity to the ligand-binding site may play a key role in ligand recognition. The observed functional studies and their implication for ligand recognition by TLRs could lead to the development of adjuvants that specifically bind to the TLR ECD and activate the TLRs or anti-inflammatory drugs that block TLR mediated signaling.
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ACKNOWLEDGEMENTS ........................................................................................................................................ I
SUMMARY ................................................................................................................................................................. II
1. INDRODUCTION .................................................................................................................................................. 1
2. TOLL-LIKE RECEPTORS
2.1 THE IMMUNE SYSTEM AND TASK OF DEFENCE ................................................................................................................................. 6
2.2 TLR SIGNALING .......................................................................................................................................................................................... 8
2.3 LEUCINE-RICH REPEATS ？ THE BUILDING BLOCKS OF TLRs .......................................................................................................... 9
2.4 STRUCTURE OF EXTRACELLULAR DOMAINS OF TLR AND THEIR LIGANDS ........................................................................... 14
2.4.1 TLR1 AND 7 SUBFAMILY ..................................................................................................................................................................... 17
3. PROTEIN STRUCTURE PREDICTION
3.1 OVERVIEW ................................................................................................................................................................................................. 20
3.2 FOUR LEVELS OF PROTEIN STRUCTURE ........................................................................................................................................... 20
3.3 PROTEIN STRUCTURE PREDICTION THEORIES ................................................................................................................................ 21
3.4 MOLECULAR DOCKING ......................................................................................................................................................................... 24
4. METHODS
4.1 MOLECULAR MODELING ...................................................................................................................................................................... 26
4.2 PROTEIN MODEL EVALUATION ............................................................................................................................................................ 27
4.3 DOCKING ................................................................................................................................................................................................... 28
4.3.1 PROTEIN-PROTEIN DOCKING ........................................................................................................................................................... 28
4.3.2 PROTEIN-LIGAND DOCKING ............................................................................................................................................................. 29
4.4 MOLECULAR DYNAMIC SIMULATION ............................................................................................................................................... 31
4.5 COMPUTATION MUTAGENSIS ANALYSIS ........................................................................................................................................... 31
4.6 MODEL VISUALIZATION AND ANALYSIS .......................................................................................................................................... 32
5. RESULTS (EXTENDED ABSTRACTS OF MANUSCRIPTS)
5.1 PAPER 1:
MOLECULAR MODELING-BASED EVALUATION OF hTLR10 AND INDETIFICATION OF POTENTIAL LIGANDS IN
TOLL-LIKE RECEPTORS SIGNALING ............................................................................................................................................................ 34
5.2 PAPER 2:
COMPARATIVE ANALYSIS OF SPECIES-SPECIFIC LIGAND RECOGNITION IN TOLL-LIKE RECEPTOR 8
SIGNALING: A HYPOTHESIS ........................................................................................................................................................................... 37
5.3 PAPER 3:
IN SILICO APPROACH TO INHIBITION OF SIGNALING PATHWAYS OF TOLL-LIKE RECEPTORS 2 AND 4 BY ST2L ................... 39
5.4 PAPER 4:
MOLECULAR MODELING-BASED EVAULATION OF DUAL FUNCTION OF IkB？ ANKYRIN REPEAT DOMAIN IN TOLL-LIKE
RECEPTOR SIGNALING ................................................................................................................................................................................... 40
6. CONCLUSIONS ................................................................................................................................................... 41
REFERENCES .......................................................................................................................................................... 43
APPENDIX
PAPER 1 .............................................................................................................................................................................................................. 46
PAPER 2 .............................................................................................................................................................................................................. 60
PAPER 3 .............................................................................................................................................................................................................. 76
PAPER 4 .............................................................................................................................................................................................................. 94
SUPPORTING INFORMATION .......................................................................................................................... 106
