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

相互作用头部基序(IHM)是肌球蛋白头部在松弛的粗丝中的一种构型 肌肉，其中ATP的周转和肌动蛋白的结合被两个头部的相互作用所抑制。在……里面 这是该领域的一个戏剧性发展，这一主题已成为公认的正常的基本特征 肌肉的松弛和收缩，通过它对粗丝活动的调节。除了在厚重中的作用 在几乎所有类型的动物中，IHM也是肌球蛋白II单分子结构的基础 手机。在这种单体形式中，肌球蛋白的尾巴折叠起来，形成一个紧密的分子，其中ATPase的活性是 同样被类似的头-头相互作用所抑制。IHM似乎在身体中扮演着两个关键角色。粗体字 细丝，它有助于在肌肉放松状态下保存能量。作为单体，它的作用是 肌球蛋白的储存形式，其紧凑的形式便于运输到其细丝组装部位。这些关键因素 新的发现是这一应用的动机：我们的目标是阐明这一基本原理的结构 骨骼肌粗丝和单个肌球蛋白分子中的调控基序，从而阐明了它是如何 功能。我们将使用最先进的低温电磁和3D重建技术来完成这项工作，研究选定的 建立系统模型，并整合从每个系统获得的信息。 在目标1中，我们将使用新型低温电子显微镜确定天然粗丝中IHM的3D结构 目前正在给结构生物学带来革命性变化的技术。利用狼蛛骨骼肌丝， 已知的最稳定的物种，我们将以高于10á的分辨率确定两者之间的相互作用 头部和头部和尾部之间，创造了IHM。在所获得的见解的帮助下，我们将 确定青蛙骨骼粗丝的结构，这是脊椎动物中最稳定的细丝。我们将在此基础上 这些信息确定了(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： 肌球蛋白作为最稳定的单分子，它将提供最高的分辨率；狼蛛 肌球蛋白作为与目标1中的细丝结构的直接联系，帮助其解释；哺乳动物的肌球蛋白， 这将揭示脊椎动物骨骼肌的结构。我们还将研究可能存在的分子 头部相互作用位点发生了突变，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疾病突变影响假说 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，为收缩和放松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。

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.