Structural Comparison of Strongly-Bound Actomyosin States

描述（由申请人提供）：肌球蛋白是基于肌动蛋白的分子马达的超家族，在动物细胞中普遍存在。肌球蛋白与丝状肌动蛋白（F-肌动蛋白）的ATP水解驱动的循环相互作用涉及多种细胞内功能，包括细胞迁移和粘附;细胞器和大分子的细胞内运输和定位;信号转导;和肿瘤抑制。这些相互作用的重要性说明了由鉴定致病肌球蛋白突变，表现在发展缺陷以及心血管和神经元疾病。所有肌球蛋白与肌动蛋白相互作用的基本机制通常是保守的，但不同的肌球蛋白已经调整了它们的结构，动力学和机械特性，以优化其特定细胞作用的性能。我们假设，肌球蛋白适应的模式，对应于特定的“结构签名”，我们的目的是在这里确定使用电子冷冻显微镜和先进的计算成像技术，专注于II类肌球蛋白，在心脏系统的背景下。 自拨款开始以来，我们的研究表明，肌球蛋白环在肌动蛋白界面（环2），在核苷酸结合口袋（环1），和一个大的裂缝，划分肌动蛋白结合区的肌球蛋白是关键的结构要素，确定在水解周期中，肌球蛋白结合肌动蛋白的模式。我们的研究提供了详细的基于残基的肌动球蛋白强结合态的界面信息。我们发现，存在一个结构相关的假设应变依赖ADP释放机制在平滑肌肌球蛋白，不存在骨骼肌肌球蛋白。这种释放机制将有利于肌球蛋白设计用于高力量和缓慢收缩。最后，我们提供了一个详细的肌球蛋白V的合成能力，其中包括以前无法访问的弱结合肌动球蛋白状态的两个结构的测定，其中之一显示了在一个“向上”的位置的第一次的手臂。 在这项提案中，我们将继续开发和使用最先进的EM图像重建方法，并使用一系列专门选择的肌球蛋白和肌动蛋白来提供（a）F-肌动蛋白的高分辨率结构，（B）详细的残留水平的肌动球蛋白界面信息（c）提供与两种心肌肌球蛋白亚型（α-和β-心肌）直接相关的“结构特征”，在与家族性肥厚型心肌病相关的肌动球蛋白装配和致病突变的背景下。 心力衰竭是一个全球性的公共卫生问题，仅在美国就影响了数百万患者。心脏病的一个主要原因是家族性肥厚性心肌病（FHC），这是一种遗传性心脏病，经常导致年轻人和其他健康个体的猝死。在这里，我们建议使用先进的成像和计算技术相结合，以提供一个深入的表征分子马达密切参与正常和异常（FHC）的心脏功能。我们的研究将为靶向治疗诊断或干预提供新的视角。

DESCRIPTION (provided by applicant): Myosins are a superfamily of actin-based molecular motors, ubiquitous in animal cells. The cyclic, ATP-hydrolysis-driven interaction of myosin with filamentous actin (F-actin) has been implicated in a variety of intracellular functions, including cell migration and adhesion; intracellular transport and localization of organelles and macromolecules; signal transduction; and tumor suppression. The importance of these interactions is illustrated by the identification of disease-causing myosin mutations, manifested in development defects as well as cardiovascular and neuronal diseases. The basic mechanism by which all myosins interact with actin is generally conserved, but different myosins have tuned their structural, kinetic, and mechanical properties to optimize performance for their particular cellular role. We hypothesize that the modes in which myosins adapted, correspond to specific "structural signatures", which we aim in determining here using electron cryo-microscopy and advanced computational imaging techniques, focusing on class II myosins, in the context of the cardiac system. Since the inception of the grant, our studies showed that myosin loops at the actin interface (loop 2), at the nucleotide-binding pocket (loop 1), and a large cleft that divides the actin-binding region of myosin are key structural elements that determine the mode in which myosin binds actin during the hydrolysis cycle. Our studies provided detailed residue-based actomyosin interface information for the strong-binding states. We showed the existence of a structural correlate to the postulated strain-dependent ADP release mechanism in smooth muscle myosin that does not exist for skeletal myosin. Such a release mechanism would benefit a myosin designed for high forces and slow contractions. Finally, we provided a detailed structural mechanism for myosin V processivity which included the determination of two structures of previously inaccessible weakly-bound actomyosin states one of which shows the lever-arm in an 'up' position for the first time. In this proposal, we will continue developing and using state-of-the-art EM image reconstruction approaches and use an array of specifically selected myosins and actins to provide (a) high-resolution structure of F-actin, (b) a detailed, residue-level actomyosin interface information (c) provide "structural signatures" directly associated with the two cardiac myosin isoforms, alpha- and beta-cardiac, in the context of the actomyosin assembly and disease-causing mutations associated with familial hypertrophic cardiomyopathy. PUBLIC HEALTH REVELANCE: Heart failure is a world wide public health problem that affects several million patients in the United States alone. One prime cause of heart disease is familial hypertrophic cardiomyopathy (FHC), which is an inherited cardiac disease that frequently results in sudden death of young and otherwise healthy individuals. Here, we propose using a combination of advanced imaging and computational techniques to provide an in-depth characterization of the molecular motors intimately involved in normal and aberrant (FHC) heart function. Our studies will provide new perspectives to consider for targeted therapeutic diagnostics or intervention.