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Structural Insights into the Protein-Ligand and Protein-DNA Interaction to Understand the Signaling Mechanism

초록/요약

TLR4 is a vital innate immune receptor that detects the presence of lipopolysaccharide (LPS) and mount an immune response. However, in case when LPS is of different origin, TLR4 discriminately initiates the signaling pathway. In the first study, molecular dynamics simulation (MDS) (25 ns) was has been employed to underscore the differential activation behavior of TLR4/MD2 complex when this complex detects Rhodobacter sphaeroides originated lipid A (RsLA). RsLA is equipped with 5-acyl chain lipid A, which activates TLR4 signaling in horses and hamsters, while inhibits TLR4 signaling in human and murine. MDS highlighted various features as the plausible cause of this variable response. These various features include solvent accessible surface area, conformational changes at local and global level, and the surface charge distribution of TLR4/MD2. Moreover, with time, the backbone of GlcN1-GlcN2 acquire the agonistic and antagonistic conformations under the influence of MD2 in horse/hamster and human/murine respectively. The contribution of various individual amino acids has also been quantified. Altogether, this study furnishes novel insights of TLR4/MD2 complex activation. In second part, the functional loss of TLR4 in response to two mutations (D299G, and T399I) has been studied. TLR4, extracellular cell surface receptor, is a conserved but variable molecule among species. However, these two mutations can abolish the TLR4 activity altogether. X-ray crystallography has provided the structural basis, however, those structural distortions should not completely block the activity. That prompted to study the dynamic behavior of TLR4/MD2 complex. MDS analysis revealed the differential number of H-bonds between protein-ligand and protein-protein molecules. Secondary structure was largely preserved, however, minor fluctuations were evident. A lower number density values, higher mean Cα distance, and substantial differences in the dihedral distribution were observed in mutated forms. Normal mode analysis (NMA) further revealed that the mutational variants of TLR4 acquired ‘z-shaped’ forms. Therefore, MDS analysis significantly illuminated the mutant-specific conformational alterations, and these are helpful in deciphering the mechanism of loss-of-function mutations. In third project, the structural features that are being perpetuated in DNA when OCT4 and SOX2 binds their cognate enhancer region during induced pluripotent stem cell generation (iPSCs) has been study. By performing extensive simulations, it has been observed that SOX2 not only bend the DNA, but also twist the DNA conformation into A-like DNA. In contrast, OCT4 binding could not influence the conformation of DNA substantially. Moreover, dynamics cross-correlation matrix of DNA heavy atoms was substantially different along with conformational entropy score. Essential dynamic analysis also revealed particular and characteristics conformational orientations and directions in response to SOX2 and/or OCT4 binding. The diffusion of DNA heavy atoms and dipole moment were also studied. These analyses showed that diffusion coefficient was lower for DNA with two proteins, and same the case for dipole moment. Taken together, our results establish a link between protein-protein and protein-DNA interactions, which may help to understand various phenomena attributed to transcriptional efficiency.

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목차

CHAPTER 1
Introduction To TLRs
1
1.1 Introduction to Toll-like Receptors (TLRs)…………… 2
1.2 TLR signaling pathway…………………………………… 3
1.3 Importance of TLRs in immune system……………… 4
1.4 An overview of literature ………………………………… 6
1.5 Conclusion and future prospects ……………………… 9
1.6 References ………………………………………………… 10
CHAPTER 2
Insights Into The Species-Specific TLR4 Signaling Mechanism In Response To Rhodobacter Sphaeroides Lipid A Detection 12
2.1 Overview of research paper………………………………13
2.2 Importance of the study………………………………… 14
2.5 References………………………………………………. 18
CHAPTER 3
Complex-Proficient While Being Signaling-Deficient: Insight Into The Effects Of Mutations On Tlr4 Function 20
3.1 Abstract……………………………………………………. 21
3.2 Introduction ……………………………………… 22
3.3 Method……………………………………………………… 23
3.4 Results……………………………………………………… 26
3.5 Discussion………………………………………………… 32
3.6 References………………………………………………… 38
CHAPTER 4
Induced Pluripotency Stem Cells (iPSCs) 45
4.1 Induced pluripotent stem cells ………………………… 46
4.2 Challenges in iPSCs …………………………………… 47
4.3 Mechanism of SOX2/OCT4 binding and its potential significance....................................................... 48

4.4 An overview of computational studies……………… 49
4.5 Conclusion and future challenges……………………… 51
4.6 References………………………………………………… 52
CHAPTER 5
Structural And Conformational Insights Into The SOX2/OCT4-Bound Enhancer DNA: A Computational Perspective 55
5.1 Abstract ……………………………………………………56
5.2 Introduction ……………………………………………… 57
5.3 Results……………………………………………………… 60
5.4 Discussion .………………………………………………… 70
5.5 Method …………………………………………………… 76
5.6 References………………………………………………… 79
6 Appendix ...………………………………………………… 93
6.1 Appendix 1 ………………………………………………… 93
6.2 Appendix 2 ………………………………………………… 104
6.3 Appendix 3 ………………………………………………… 107

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