Structural Comparison of Strongly-Bound Actomyosin States

描述(申请人提供)：肌球蛋白是一个基于肌动蛋白的分子马达超家族，广泛存在于动物细胞中。肌球蛋白与丝状肌动蛋白(F-肌动蛋白)的相互作用在细胞内具有多种功能，包括细胞迁移和黏附、细胞器和大分子在细胞内的运输和定位、信号转导和肿瘤抑制等。这些相互作用的重要性通过鉴定致病肌球蛋白突变来说明，这些突变表现在发育缺陷以及心血管和神经疾病上。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、动力学和力学性质，以优化其特定的细胞角色的性能。我们假设肌球蛋白适应的模式对应于特定的“结构特征”，我们的目标是在心脏系统中使用电子冷冻显微镜和先进的计算机成像技术来确定这些特征，重点是II类肌球蛋白。 自拨款开始以来，我们的研究表明，肌球蛋白在肌动蛋白界面(环2)、核苷酸结合口袋(环1)以及将肌球蛋白的肌动蛋白结合区分开的大裂隙是决定肌球蛋白在水解循环中结合肌动蛋白的方式的关键结构元件。我们的研究为强结合态提供了详细的基于残基的肌动球蛋白界面信息。我们发现，在肌球蛋白中存在一种结构上与假定的应变依赖的ADP释放机制相关，而骨骼肌球蛋白不存在这种机制。这样的释放机制将有利于设计用于高力和慢收缩的肌球蛋白。最后，我们提供了肌球蛋白V过程的详细结构机制，包括确定了两种以前无法获得的弱结合肌球蛋白状态，其中一种结构首次显示杠杆臂处于“上”位置。 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，并使用一系列特定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、残基水平的肌球蛋白界面信息；(C)在肌动球蛋白组装和与家族性肥厚性心肌病相关的致病突变的背景下，提供与两种心肌肌球蛋白亚型(α-和β-心脏)直接相关的“结构特征”。 公共卫生评论：心力衰竭是一个世界性的公共卫生问题，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，经常导致年轻的和其他健康的人突然死亡。在这里，我们建议使用先进的成像和计算技术的组合来提供与正常和异常(FHC)心脏功能密切相关的分子马达的深入表征。我们的研究将为有针对性的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.