Coupling between conformation and chemistry in enzymes

描述(申请人提供)：肌球蛋白是一个典型的分子马达超家族，在囊泡运输、细胞运动、肌肉收缩和信号转导等多种生物学过程中发挥重要作用。虽然肌球蛋白的功能循环是以离线的形式被理解的，但关于运动域的构象特性和ATPase活性之间的耦合仍然有许多详细的问题。 我们假设肌球蛋白门ATP水解过程中的构象转变不仅通过调节活性部位的特定氨基酸的位置，而且还通过调节水解点周围水分子的方向和动力学来实现。为了验证和巩固这一假说，提出了最新的分子模拟来分析肌球蛋白II马达结构域和相关突变体不同构象状态下的ATP水解机制，这些模拟包括经典的分子动力学方法和QM/MM组合方法。具体目的是：(1)确定运动结构域封闭状态下的ATP水解酶催化机理。(2)确定在运动域的开放状态下是否禁止ATP水解，如果是，识别决定水解能的开放构象和闭合构象之间的关键差异。(3)从能量学和机械学的角度解释活性位点残基的作用，诱变研究表明活性残基对ATP的水解性和运动性有不同的影响。 之所以选择肌球蛋白-11，是因为它是唯一一个具有多种构象状态的高分辨率结构的马达系统，计算结果可以与大量的生化和生物物理数据进行比较。拟议的模拟研究将提供一个框架，用于连接来自不同学科的实验数据，以建立肌球蛋白和其他分子马达中机械力化学耦合的合理理论模型；微观洞察将对我们设计治疗由肌球蛋白功能障碍引起的严重疾病(如心肌病)的策略的能力产生深远影响。 模拟工作将通过合作与实验研究紧密结合；结构、动力学和运动学数据的结合将提供验证和完善模拟技术所需的实验测试，这对计算酶学领域具有巨大价值。

DESCRIPTION (provided by applicant): Myosin is a superfamily of prototypical molecular motors that play important roles in diverse biological processes ranging from vesicle trafficking, cell motility to muscle contractions and signal transductions. Although the functional cycle of myosins is understood in an out-line form, many detailed questions remain concerning the coupling between conformational properties of the motor domain and the ATPase activity. We hypothesize that conformational transitions in myosin gate ATP hydrolysis through regulating not only positions of specific amino acids in the active site but also the orientation and dynamics of water molecules surrounding the hydrolysis site. To verify and consolidate such a hypothesis, state-of-the-art molecular simulations are proposed to analyze the mechanism of ATP hydrolysis in different conformational states of the myosin II motor domain and relevant mutants; the simulations include classical molecular dynamics and combined QM/MM methods. The specific aims are: (1) Determine the catalytic mechanism of ATP hydrolysis in the closed state of the motor domain. (2) Determine if ATP hydrolysis is prohibited in the open state of the motor domain, and if so, identify key differences between the open and closed conformations that dictate the hydrolysis energetics. (3) Explain, in energetical and mechanistic terms, the roles of active site residues, which have been shown by mutagenesis studies to have various effects on ATP hydrolysis and motility. Myosin-ll was chosen because it is the only motor system that has high-resolution structures for multiple conformational states, and computational results can be compared with a large body of biochemical and biophysical data. The proposed simulation study will provide a framework for bridging experimental data from different disciplines to establish sensible theoretical models for mechanochemical coupling in myosin and other molecular motors; the microscopic insights will have a profound impact on our ability to design strategies to treating serious diseases caused by myosin dysfunction such as cardiomyopathy. The simulation work will be closely coupled to experimental studies through collaborations; the combination of structural, kinetic and motility data will provide the experimental tests necessary to verify and refine simulation techniques, which is of tremendous value to the field of computational enzymology.