Structural Comparison of Strongly-Bound Actomyosin States

强结合肌动球蛋白状态的结构比较

• 批准号: 7932528
• 负责人: DORIT HANEIN
• 金额: $ 21.49万
• 依托单位: SANFORD BURNHAM PREBYS MEDICAL DISCOVERY INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-30 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7932528
• 关键词: ATP Hydrolysis; Actins; Actomyosin; Adhesions; Affect; Algorithms; Amino Acids; Anatomy; Animals; Arts; Binding; Biochemical; Bioinformatics; Biological Models; Cardiac; Cardiac Myosins; Cardiovascular system; Cell physiology; Cells; Characteristics; Cleaved cell; Communities; Complex; Computational Technique; Cryoelectron Microscopy; Data; Data Collection; Defect; Deoxyribonucleases; Development; Diagnostic; Disease; Docking; Electron Microscopy; Elements; Event; F-Actin; Familial Hypertrophic Cardiomyopathy; Funding; Gel; Glutamine; Goals; Grant; Hand; Head; Heart Diseases; Heart failure; Hemoglobin; Histology; Histopathology; Hydrolysis; Image; Image Analysis; Imaging Techniques; Imaging technology; Individual; Inherited; Intervention; Intracellular Transport; Kinetics; Knowledge; Laboratories; Light; Link; Mammals; Maps; Mechanics; Medical; MgADP; Modeling; Molecular; Molecular Conformation; Molecular Machines; Molecular Motors; Motor; Movement; Mus; Muscle; Mutation; Myofibrils; Myosin ATPase; Myosin Type II; Myosin Type V; NMR Spectroscopy; Neurons; Nucleotides; Organ; Organelles; Output; Pathology; Patients; Performance; Phase; Phenotype; Physiological; Point Mutation; Positioning Attribute; Preparation; Property; Protein Isoforms; Public Health; Rattus; Research; Research Personnel; Resolution; Role; Sampling; Science; Sequence Homology; Sickle Cell Anemia; Signal Transduction; Skeletal Muscle; Skeletal Muscle Myosins; Slide; Smooth Muscle; Smooth Muscle Myosins; Specificity; Structure; Sudden Death; Surface; System; Technology; Testing; Time; Transgenic Mice; Tumor Suppression; United States; Upper arm; Ventricular Cardiac alpha-Myosin; Vertebral column; Work; X ray diffraction analysis; X-Ray Crystallography; X-Ray Diffraction; base; beta pleated sheet; beta-Myosin; cell motility; design; disease-causing mutation; gain of function; heart function; human disease; image reconstruction; improved; macromolecule; molecular assembly/self assembly; monomer; mutant; protein expression; reconstruction; response; skeletal; therapeutic target; three dimensional structure; tool

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.

描述（由申请人提供）：肌醇是基于肌动蛋白的分子电机的超家族，在动物细胞中无处不在。肌球蛋白与丝状肌动蛋白（F-肌动蛋白）的循环，ATP-Hydrolsosy驱动的相互作用与各种细胞内功能有关，包括细胞迁移和粘附。细胞内运输和细胞器和大分子的定位；信号转导；和抑制肿瘤。这些相互作用的重要性通过鉴定引起疾病的肌球蛋白突变的鉴定来说明，该突变体现在发育缺陷以及心血管和神经元疾病中。所有肌球蛋白与肌动蛋白相互作用的基本机制通常都是保守的，但是不同的肌动物调整了它们的结构，动力学和机械性能，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“结构特征”，我们旨在在这里使用电子冷冻微观显微镜和先进的计算成像技术来确定，在心脏系统的背景下，它重点介绍了II类肌球蛋白。 自赠款成立以来，我们的研究表明，肌动蛋白界面处的肌球蛋白环（环2），核苷酸结合口袋（环1）和大裂口，肌球蛋白的肌动蛋白结合区域是关键结构元素，是确定肌球蛋白在水解周期内结合肌动蛋白的模式的关键结构元素。我们的研究为强结合状态提供了详细的基于残余的肌动蛋白界面信息。我们展示了与平滑肌肌球蛋白中假定的菌株依赖性ADP释放机制相关的存在，而骨骼肌球蛋白不存在。这样的释放机制将使用于高力和缓慢收缩的肌球蛋白受益。最后，我们为肌球蛋白V加工率提供了详细的结构机制，其中包括确定以前无法访问的弱结合的肌动蛋白状态的两个结构，其中一种显示了第一次“向上”位置的杠杆臂。 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与家族性心肌病有关的肌动球蛋白组装和引起疾病的突变。 公共卫生的启示：心力衰竭是一个全球公共卫生问题，仅在美国就会影响数百万患者。心脏病的一个主要原因是家族性肥厚性心肌病（FHC），它是一种遗传性心脏病，经常导致年轻人和其他健康的个体突然死亡。在这里，我们建议使用先进的成像和计算技术的组合，以提供与正常和异常（FHC）心脏功能密切相关的分子电机的深入表征。我们的研究将为有针对性的治疗诊断或干预提供新的观点。