Dissecting the structural origin of relaxation in skeletal muscle

肌肉如何收缩一直是一个长期存在的问题。尽管这一领域取得了重大进展，但如何 肌肉松弛仍未完全了解。收缩是由于含有肌球蛋白的厚浆滑动而发生的 含有肌动蛋白的细丝，由肌球蛋白头部提供动力，由三磷酸腺苷提供燃料，产生滑动力。 当细丝被切断，磁头不能绑定以产生力时，就会发生松弛，使空转 头部在粗大的丝状物中螺旋排列。目前对Thick的作用已知多少？ 长丝在放松吗？在结构方面，心脏(小鼠、人)和骨骼的低分辨率模型 (狼蛛)已经获得了粗丝，但它们的原子结构仍未解决。关于能量学 另一方面，放松骨骼肌的能量消耗揭示了一个令人惊讶的现象，即所谓的超级 放松(SRX)，大大减少了ATP的消耗。一种被广泛接受的观点将这种无处不在的 基本的节能状态，在放松的狼蛛中肌球蛋白的两个头折叠在一起的独特方式 细丝--所谓的相互作用头部基序(IHM)，发现于动物界，在结构上 抑制两个头部，关闭它们的活动。不管它的吸引力如何，这个SRX=IHM假说并没有 事实证明，最近的ATP周转结果表明，SRX与特定的肌球蛋白头部有关 构象。阐明这个谜题对于理解肌肉是如何松弛的，它是如何发生故障的 以及治疗性药物治疗是如何起作用的。溶液需要用低温电子显微镜测定原子 肌肉中粗丝和肌球蛋白分子的结构。在这里，我们建议确定结构 骨骼肌球蛋白分子和细丝，远比心脏研究得少。这将使我们能够剖析 IHM的相互作用限制了两个头部的活动，关闭了它们，从而使ATP保持在松弛状态。我们会 用单粒子EM和低温EM确定厚层中近原子水平的SRX态的结构基础 兔骨骼肌细丝和肌球蛋白分子。通过与狼蛛进行比较，显示出十倍于- 更节能(超松弛，HRX)，我们将对ATPase的机制有更深入的了解 抑制力。我们将使用EM和X射线衍射来研究治疗药物如何改变IHM。 目标1将通过揭示其近原子结构来定义骨骼粗丝中SRX的结构基础 低温电磁结构。目标2将确定骨骼肌球蛋白头和重肌球蛋白中SRX的结构基础 通过评估：(A)SRX是否来自特定的头部构象，以及(B)IHM是否与 SRX状态。目标3将揭示药物对骨骼粗丝和肌球蛋白分子的结构影响。 尽管SRX在骨骼肌松弛中起着重要作用，但其结构基础及其与IHM和IHM的关系 对其他粗丝蛋白(MyBP-C、Titin)的作用尚不清楚。我们的研究将揭示IHM的结构 骨骼粗丝和肌球蛋白分子，阐明其与SRX状态和MyBP-C和 Titin，并对松弛的分子基础和治疗药物的影响提供关键的见解。

How muscle contracts has been a long-standing question. Despite major advances in this area, how muscle relaxes is still not fully understood. Contraction occurs by the sliding of myosin-containing thick past actin-containing thin filaments, powered by myosin heads, motors that produce sliding force, fueled by ATP. Relaxation occurs when thin filaments are switched off so heads cannot bind to produce force, leaving the idling heads to organize themselves helically in the thick filament. What is currently known about the role of thick filaments in relaxation? On the structural side, low-resolution models of cardiac (mouse, human) and skeletal (tarantula) thick filaments have been achieved, but their atomic structure remains unsolved. On the energetics side, the energy consumption of relaxed skeletal muscle revealed a surprising phenomenon, so-called super- relaxation (SRX) that greatly reduces ATP consumption. A widely accepted view associates this ubiquitous and fundamental energy-saving state with the unique way myosin’s two heads fold together in the relaxed tarantula filament—the so-called interacting-heads motif (IHM), found across the animal kingdom, which structurally inhibits both heads, switching off their activity. Regardless of its appeal, this SRX=IHM hypothesis has not been proved, and recent ATP turnover results suggest, instead, association of SRX with a specific myosin head conformation. Elucidating this puzzle is crucial to understanding how muscle relaxes, how it malfunctions in disease and how therapeutic drug treatments work. The solution requires determination by cryo-EM of the atomic structures of the thick filament and myosin molecules from muscle. Here, we propose to determine the structures of skeletal myosin molecules and filaments, far less studied than cardiac. This will allow us to dissect how key IHM interactions constrain activity of the two heads, shutting them off, thus conserving ATP in relaxation. We will use single particle EM and cryo-EM to define the structural basis of the SRX state at near-atomic level in thick filaments and myosin molecules from rabbit skeletal muscle. By comparing with tarantula, which shows tenfold- greater energy-saving (hyper-relaxation, HRX), we will gain deeper insight into the mechanism of ATPase inhibition. And we will use EM and X-ray diffraction to investigate how therapeutic drugs alter the IHM. Aim 1 will define the structural basis of SRX in skeletal thick filaments by revealing their near-atomic cryo-EM structures. Aim 2 will define the structural basis of SRX in skeletal myosin heads and heavy meromyosin molecules by assessing: (A) if SRX results from a specific head conformation, and (B) if the IHM correlates with the SRX state. Aim 3 will reveal the structural impact of drugs on skeletal thick filaments and myosin molecules. Despite the vital role of SRX in skeletal muscle relaxation, its structural basis and relation to the IHM and to other thick filament proteins (MyBP-C, titin) remains unknown. Our studies will reveal the IHM structure in skeletal thick filaments and myosin molecules, clarify its association with the SRX state and with MyBP-C and titin, and provide critical insights into the molecular basis of relaxation and the influence of therapeutic drugs.