Towards Understanding Toll-like Receptor Structure and Function through Biomolecular Simulations
- 주제(키워드) Bioinformatics
- 발행기관 아주대학교
- 지도교수 Sangdun Choi
- 발행년도 2020
- 학위수여년월 2020. 8
- 학위명 박사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000030213
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Toll-like receptors (TLRs) constitute a superfamily of pattern recognition receptors (PRRs), which recognize pathogen-associated molecular patterns (PAMPs) or host-derived damage-associated molecular patterns (DAMPs) and initiate an immune cell response, primarily through macrophages and dendritic cells. These receptors are type-I transmembrane (TM) proteins, consisting of an extracellular domain (ECD), a single-pass TM domain, and a cytosolic Toll/interleukin-1 receptor (TIR) domain. Upon activation they trigger a complex cascade of signal transduction to induce the expression of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and antiviral interferons (IFNs). TLRs and their downstream adaptors, such as, TIR domain-containing adaptor proteins (TIRAP), are multidomain proteins and form either dimerization or oligomerization for executing their function. Owing to the advancement of structural biology techniques, such as X-ray crystallography or NMR spectroscopy, experimental structures of TLR individual domain and their adaptors are publicly available. However, currently accessible TLR-related structural data provide us with limited understanding of the complex mechanism of receptor activation and membrane localization. Therefore, the structure of all-domain, full-length proteins in presence of their membrane-aqueous environment are essential for obtaining an in-depth knowledge of the receptor/adaptor organization for the development of novel therapeutic modulators. To bridge this knowledge-gap associated with the structure of TLRs and their adaptor proteins, I used comparative protein modeling in combination with molecular dynamics (MD) simulations considering model phospholipid bilayers to obtain a comprehensive idea about the membrane integration and dynamics of TLRs. First, I constructed an all-residue model of TLR4, which is a well-studied member of TLR superfamily, using homology modeling, protein-protein docking, and MD simulations. The conformational dynamics of all three domains of TLR4 seemed biophysically relevant for its integration into the cell membrane. The ECD and TIR domains were partially absorbed into the hydrophilic regions of the bilayer; meanwhile the TM domains showed a considerable tilt because of the hydrophobic mismatch with the bilayer core. The TLR4-TM domain could possibly form an alternate homodimeriztion of oligomerization assembly in addition to the canonical interface. The exposed and buried surfaces of the TIR microdomains suggested the most-likely surfaces for the adaptor recruitment. The intact signaling competent form of TLR4 in the lipid bilayer can be helpful in the design of novel modulators to activate or inhibit the TLR4-dependent immune responses. Second, I investigated an all-domain TLR3 structure inside a model phospholipid bilayer using MD simulations. A ~30-35° tilt of TLR3-ECD was noticed on the membrane surface due to the asymmetrical electrostatic interactions with phospholipid headgroups. The sugar-phosphate backbone of dsRNA did not exhibit a significant conformational changes but the TM domain showed curvature and large crossing angle to avoid the hydrophobic mismatch with the bilayer core. The TIR domains were partially absorbed into the lower leaflet of the bilayer by means of the helix αD and the CD and DE loops. Based on the simulation data, the reciprocal interaction between αC and αD helices of one subunit and the helix αC and the BB loop of the other could be the homodimerization interface of the TLR3-TIR. Altogether, this work increases our knowledge about the activation and membrane-organization of TLR3 in a physiological environment. Third, I studied an all-residue model of TIRAP dimer and investigated its interaction with the phosphatidylinositol (PI) molecules, PIP2 and PIP3, which are important components of the cell/endosomal membrane. Results indicated that PIP2 provide the membrane with a stable microdomain for TIRAP binding in a physiologically relevant orientation. The binding free energy of the PI-binding domain (PBD) of TIRAP for PIP2 was found to be highly essential for its anchoring to the membrane. At least, four PIP2 molecules are required by the lysine-rich surface of the PIP2-binding motif. Substitution of alanine in place of the PI-binding residues had reduced the overall affinity of TIRAP for PIP2. Three previously unknown positively charged residues K34, K35, and R36 were predicted to be crucial for the increased affinity of TIRAP towards PIP2. Among these residues, R36 interacted with PIP2 through consistent hydrogen bonds and van der Waals interaction. Similarly, PIP3 interacted with TIRAP in an analogous manner through an H-bond network involving K34, K35, and R36. This work redefine our current understanding of the membrane-anchoring properties of TIRAP and could be useful for the design of novel decoy peptides for modulating TLR-dependent immune responses. Conclusively, the results from the explicit membrane MD simulation of full-length TLR3, TLR4, and TIRAP is deemed helpful in designing novel peptide/small molecule agents to modulate the TLR-mediated immune exacerbation.
more목차
1. Introduction 1
1.1. Toll-like receptors (TLRs) 2
1.2. The signaling pathways of TLRs 4
1.3. Therapeutic significance of TLRs 6
1.4. The overall structural topology of TLRs 7
2. Methods 9
2.1. Construction of a full-length, active-state model of TLR4 10
2.2. Solvation of phospholipid molecules around TLR4 10
2.3. MD simulation parameters for TLR4-membrane system 11
2.4. MD simulations of TLR4-TIR dimers in water 11
2.5. MD simulation of TLR4-TM domain in the phospholipid bilayer 12
2.6. Docking of TAK-242 with TLR4-TIR models 12
2.7. Construction of full-length, active-state models of TLR3 12
2.8. Solvation of phospholipids around TLR3 13
2.9. MD simulation parameters for TLR3-membrane systems 13
2.10. Construction of the full-length active-state model of TIRAP 13
2.11. MD simulations of TIRAP-membrane systems 14
2.12. Principal component analysis of TIRAP MD trajectory 14
2.13. Binding free-energy calculation 15
2.14. Calculation of the Free energy landscape 15
2.15. Electrostatic potential surface 16
2.16. Validation of modeled proteins 16
2.17. Protein structure network 16
2.18. Miscellaneous properties 16
3. Results and Discussion (1) 17
3.1. Summary 19
3.2. Results 20
3.3. Discussion 33
3.4. Conclusion 37
4. Results and Discussion (2) 38
4.1. Summary 40
4.2. Results 41
4.3. Discussion 57
4.4. Conclusion 61
5. Results and Discussion (3) 62
5.1. Summary 64
5.2. Results 66
5.3. Discussion 79
5.4. Conclusion 82
6. References 83
7. Appendix 92
