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.

相互作用头基序（IHM）是松弛的粗丝中肌球蛋白头的配置 肌肉中 ATP 周转和肌动蛋白结合受到每个头部与另一个头部相互作用的抑制。在 这一领域的巨大发展，这一主题已被认为是正常的基本特征 肌肉放松和收缩，通过其调节粗肌丝活动。除了它的厚重作用外 除了细丝之外，IHM 也是几乎所有类型动物中肌球蛋白 II 单分子结构的基础 细胞。在这种单体形式中，肌球蛋白尾部折叠起来，形成一个紧凑的分子，其中 ATP 酶活性为 再次受到类似的头-头相互作用的抑制。 IHM 似乎在体内发挥着两个关键作用。厚中 细丝，它有助于肌肉放松状态下的能量保存。作为单体，它的功能是 肌球蛋白的储存形式，其紧凑的形式有利于运输到其丝组装位点。这些关键的 新的发现是这个应用的动机：我们的目标是阐明这个基本的结构 骨骼肌粗丝和单个肌球蛋白分子中的调节基序，从而阐明它是如何 功能。我们将使用最先进的冷冻电镜和 3D 重建技术来做到这一点，研究选定的 模型系统并整合从每个系统获得的信息。 在目标 1 中，我们将使用新型冷冻电镜确定天然粗丝中 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， 已知最稳定的物种，我们将以优于 10 Å 的分辨率确定两者之间的相互作用 头部以及头部和尾部之间，形成 IHM。借助所获得的见解，我们将 确定青蛙骨骼粗丝的结构，是脊椎动物中最稳定的丝。我们将在此基础上继续发展 该信息可用于确定（不太稳定的）哺乳动物粗丝中 IHM 的结构。在目标 2 中，我们 将使用三个互补系统确定分离的肌球蛋白分子中 IHM 的 3D 结构： 平滑肌肌球蛋白作为最稳定的单分子，将提供最高的分辨率；狼蛛 肌球蛋白作为目标 1 中与丝结构的直接联系，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。我们还将检查假定的分子 头部相互作用位点已发生突变，以测试它们在 IHM 形成中的重要性。在目标 3 中，我们将使用 单分子 EM 检验骨骼肌球蛋白头部区域疾病突变影响的假设 IHM 的稳定性。 IHM 现在被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，为收缩和松弛的结构基础提供新的见解，以及 疾病突变对这些功能的潜在影响。