Structure of The Interacting-Heads Motif in Myosin Filaments and Molecules

肌球蛋白丝和分子中相互作用头基序的结构

• 批准号: 10189521
• 负责人: ROGER W CRAIG
• 金额: $ 40.5万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-14 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10189521
• 关键词: 3-Dimensional; ATP phosphohydrolase; Actins; Animals; Binding; Binding Proteins; Biological Models; Cardiac Myosins; Cardiomyopathies; Cells; Collaborations; Cryoelectron Microscopy; Data; Development; Disease; Electron Microscopy; Electrons; Filament; Goals; Head; Knowledge; Lead; Leg; Link; Mechanics; Methods; Microscopic; Modeling; Molecular; Motivation; Muscle; Muscle Cells; Muscle Contraction; Muscle function; Muscle relaxation phase; Mutate; Mutation; Myopathy; Myosin ATPase; Myosin Type II; Physiological; Play; Problem Solving; Proteins; Rana; Rattus; Regulation; Relaxation; Resolution; Role; Site; Skeletal Muscle; Skeletal Muscle Myosins; Smooth Muscle; Smooth Muscle Myosins; Structure; System; Tail; Techniques; Technology; Testing; Thick; Thick Filament; Thinness; Work; experience; improved; in vivo; innovation; insight; molecular imaging; monomer; muscle physiology; novel; obesity treatment; particle; reconstruction; repaired; single molecule; skeletal; structural biology; three dimensional structure

The interacting-heads motif (IHM) is a configuration of myosin heads in relaxed thick filaments of muscle, in which ATP turnover and actin binding are inhibited by the interaction of each head with the other. In a dramatic development in the field, this motif has become recognized as a fundamental feature of normal muscle relaxation and contraction, through its regulation of thick filament activity. In addition to its role in thick filaments, the IHM also underlies the structure of single molecules of myosin II in almost all types of animal cell. In this monomeric form, the myosin tail folds up, forming a compact molecule, in which ATPase activity is again inhibited by similar head-head interactions. The IHM appears to play two key roles in the body. In thick filaments, it contributes to energy conservation in the relaxed state of muscle. As a monomer, it functions as a storage form of myosin whose compact form facilitates transport to its site of filament assembly. These critical new findings are the motivation for this application: our goal is to elucidate the structure of this fundamental regulatory motif in skeletal muscle thick filaments and single myosin molecules, thus illuminating how it functions. We will do this using state-of-the-art cryo-EM and 3D reconstruction techniques, studying selected model systems and integrating the information gained from each. In Aim 1 we will determine the 3D structure of the IHM in native thick filaments using novel cryo-EM technology that is currently revolutionizing structural biology. Using tarantula skeletal muscle filaments, the most stable species known, we will determine at better than 10 Å resolution the interactions between the two heads, and between the heads and the tail, that create the IHM. With help from the insights gained, we will determine the structure of frog skeletal thick filaments, the most stable vertebrate filament. And we will build on this information to determine the structure of the IHM in (less stable) mammalian thick filaments. In Aim 2, we will determine the 3D structure of the IHM in isolated myosin molecules, using three complementary systems: smooth muscle myosin as the most stable single molecule, which will provide the highest resolution; tarantula myosin as a direct link to the filament structure in Aim 1, aiding its interpretation; and mammalian myosin, which will reveal the structure in vertebrate skeletal muscle. We will also examine molecules in which putative head interaction sites have been mutated, to test their importance in formation of the IHM. In Aim 3, we will use single molecule EM to test the hypothesis that disease mutations in the head region of skeletal myosin impact the stability of the IHM. The IHM is now recognized as a fundamental motif of normal muscle function. Our proposal will elucidate its interactions, providing new insights into the structural basis of contraction and relaxation, and of the potential impact of disease mutations on these functions.

相互作用的头主题（IHM）是肌球蛋白头的构型 肌肉，其中每个头部与彼此的相互作用抑制了ATP的周转率和肌动蛋白结合。在 该田间的戏剧性发展，该主题已被公认为是正常的基本特征 肌肉放松和收缩，通过调节厚丝活性。除了其在厚度中的作用 细丝，IHM还基于几乎所有类型的动物的肌球蛋白II分子的结构 细胞。在这种单体形式中，肌球蛋白尾巴折叠起来，形成一个紧凑的分子，其中ATPase活性为 再次被类似的头头相互作用抑制。 IHM似乎在体内扮演两个关键角色。厚 细丝，它有助于在肌肉松弛状态下节能。作为单体，它起作用 肌球蛋白的储存形式的紧凑形式有助于运输到其细丝组件的位置。这些关键 新发现是此应用的动力：我们的目标是阐明这种基本的结构 骨骼肌厚细丝和单个肌球蛋白分子的调节基序，从而阐明了它的方式 功能。我们将使用最先进的冷冻EM和3D重建技术来进行此操作，研究选定的 模型系统并整合从每个信息中获得的信息。 在AIM 1中，我们将使用新型的Cryo-EM确定本机厚细丝中IHM的3D结构 目前正在彻底改变结构生物学的技术。使用狼蛛骨骼肌丝， 最稳定的物种已知，我们将确定两者之间的相互作用比10Å分辨率更好 头部以及在头部和尾巴之间创建IHM。在获得的见解的帮助下，我们将 确定青蛙骨骼厚细丝的结构，最稳定的脊椎动物细丝。我们将基础 这些信息以确定（不稳定的）哺乳动物厚细丝中IHM的结构。在AIM 2中，我们 将使用三个完整的系统确定IHM中IHM的3D结构： 平滑肌肌球蛋白是最稳定的单分子，将提供最高的分辨率；狼蛛 肌球蛋白作为与AIM 1中细丝结构的直接联系，有助于其解释；和哺乳动物肌球蛋白， 这将揭示脊椎动物骨骼肌的结构。我们还将检查推定的分子 头部相互作用位点已突变，以测试其在IHM形成中的重要性。在AIM 3中，我们将使用 单分子EM检验以下假设：骨骼肌球蛋白撞击头部疾病突变 IHM的稳定性。 现在，IHM被公认为是正常肌肉功能的基本基序。我们的建议将 阐明其相互作用，为收缩和放松的结构基础提供新的见解，并提供 疾病突变对这些功能的潜在影响。