Dynamics Research On The Gating Mechanism Of Nicotinic Acetylcholine Receptor

Since molecular dynamics (MD) method was first performed on the protein molecule in 1970's, MD simulation has become more and more popular in biological science fields particularly. It provides a way in silico to study the intra- and inter-molecular reaction dynamically at atomic level. To date not only the scale of simulation system keeps increasing rapidly, but also the developments have been gained in MD method itself. For example, steered molecular dynamics (SMD) as a branch of MD plays an important role in the research of ligand binding/unbinding process after the late 1990's. There are three related workings presented in this dissertation. Firstly, the mechanism of the nicotinic acetylcholine receptor (nAChR) gating is investigated by a long-time (30-ns) conventional MD (CMD) simulation. Furthermore, to address the motions occurred in the physiology timescale a novel steered rotation MD (SRMD) is developed. Secondy, to further study the cause of rotation of the nAChR extracellular (EC) domain revealed in the previous work, SMD simulations are carried out to the acetylcholine binding protein (AChBP), which is an unique surrogate for the EC domain of nAChR, using three pulling models designed by us, respectively. At last, an improved SMD method is proposed and a comparison between the conventional SMD and the improved SMD reveals the advantages of the latter by applying to dissociate the cytochrome P450 3A4-metyrapone complex.The dissertation is made up of five chapters, which is summarized as follows:In Chapter I, the actuality and basic concepts of MD method, as well as ligand-gated ion channel are introduced. In additional, the main working and finding in the dissertation are briefly presented.In Chapter II, as preliminaries, introduces the theoretical and applied aspects of MD. At first, a brief history of MD is reviewed and the principles of Newton's equation of motion, the inter-atom potential function and the finite difference method are referred. Introductions on SMD method and free energy calculation are also involved here. Then the causes of using and developing MD are summed up and the introduction of the force field's development is involved as well as the MD algorithms. In the last section of this chapter, the achievements of MD in the biological science field are reviewed and the further of MD performed on biological macromolecules are predicted.The nAChR is a key molecule involved in the propagation of signals in the central nervous system and peripheral synapses. Although numerous computational and experimental studies have been performed on this receptor, the structural dynamics of the receptor underlying the gating mechanism is still unclear. In Chapter III, to address the mechanical fundamentals of nAChR gating, both CMD and SRMD simulations have been conducted on the nAChR embedded in a lipid bilayer and water molecules. To mimic the pulsive action of ACh binding, non-equilibrium MD simulations were performed by using the SRMD method developed by us. The result confirmed all the motions derived from the 30-ns CMD simulation and normal mode analysis. In addition, the SRMD simulation indicated that the channel may undergo an open-close motion. The present MD simulations explore the structural dynamics of the receptor under its gating process and provide a new insight into the gating mechanism of nAChR at the atomic level.The ligand binding/unbinding process is critical to understand the pharmacology of both the nAChR and the acetylcholine binding protein (AChBP). In Chapter IV, SMD simulations are performed to learn about the unbinding process of the full agonist nicotine. Three different pulling models are designed to investigate the possible binding/unbinding pathways: radial and tangent models, and also a mixed model. Of the three, the tangent pulling model finally fails to dissociate nicotine from the ligand binding pocket. The efficiency of the pulling force profiles is superior and the opening of the C-loop is larger in the mixed pulling model than that in the radial model. The most favorable pathway for the cholinergic agonist nicotine to enter or leave the binding pocket is through the principal binding side, following a curvilinear track. Noticeably, it has been seen that the unbinding of the nicotine is concomitant with a global rotation of the protein-ligand complex which could be caused by the interactions of the ligand with protein at the tangent direction.In Chapter V, as the finial chapter, an improved SMD method with direction optimization is proposed to dissociate ligand molecule from receptor. A multi-population genetic algorithm based on the information entropy is developed to search the optimal pulling direction. By imposing an optimization phase in the conventional SMD simulation, a better substrate-exit channel for the buried active site can be found. The novel simulation method has been used to dissociate the substrate-bound complex structure of cytochrome P450 3A4-metyrapone. The results show that the new pathway obtained by the proposed method has advantages such as lower energy barrier, less dissociation time and shorter motion trajectory than that by the conventional SMD. Additionally, the results also show that the method can dissociate successfully even along a rejected direction.