Dissecting the structural origin of relaxation in skeletal muscle

剖析骨骼肌松弛的结构起源

• 批准号: 10567284
• 负责人: Raul Padron
• 金额: $ 58.37万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-04-01 至 2028-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10567284
• 关键词: ATP phosphohydrolase; Actins; Animals; Area; Binding; Biochemical; Bone structure; Cardiac; Collaborations; Consumption; Contracts; Cryoelectron Microscopy; DNA Sequence Alteration; Disease; Drug Targeting; Energy consumption; Equilibrium; Exhibits; Fiber; Filament; H-Meromyosin; Head; Human; Impairment; Laboratories; Life; MLL gene; Measures; Microscope; Modeling; Molecular; Molecular Conformation; Molecular Motors; Motor; Movement; Mus; Muscle; Muscle Contraction; Muscle function; Muscle relaxation phase; Myopathy; Myosin ATPase; Oryctolagus cuniculus; Pharmaceutical Preparations; Pharmacotherapy; Proteins; Relaxation; Resolution; Role; Side; Skeletal Muscle; Slide; Structure; Tail; Testing; Therapeutic; Thick; Thick Filament; Thin Filament; Work; X ray diffraction analysis; antagonist; connectin; insight; particle; repaired; skeletal

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.

肌肉如何收缩一直是一个长期存在的问题，尽管在这一领域取得了重大进展，但肌肉如何收缩却是一个长期存在的问题。 肌肉放松是通过含有厚厚的肌球蛋白的滑动而发生的，目前尚不完全清楚。 含有肌动蛋白的细丝，由肌球蛋白头提供动力，肌球蛋白头是产生滑动力的马达，由 ATP 提供燃料。 当细丝关闭时，就会发生松弛，因此头部无法结合产生力，从而导致空转 目前已知粗丝的作用是什么。 在结构方面，心脏（小鼠、人类）和骨骼的低分辨率模型 （狼蛛）粗丝已经实现，但其原子结构在能量学上仍未解决。 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 一种广泛接受的观点认为，这种放松（SRX）可以大大减少 ATP 的消耗。 在放松的狼蛛中，肌球蛋白的两个头以独特的方式折叠在一起，从而达到基本的节能状态 细丝——所谓的相互作用头基序（IHM），在整个动物界都有发现，其结构 抑制两个头部，关闭它们的活动，无论其吸引力如何，这种 SRX=IHM 假设尚未得到证实。 事实证明，最近的 ATP 周转结果表明，SRX 与特定的肌球蛋白头有关联 阐明这个谜题对于理解肌肉如何放松以及它如何发生故障至关重要。 该解决方案需要通过原子冷冻电镜来确定。 在这里，我们建议确定肌肉的粗丝和肌球蛋白分子的结构。 骨骼肌球蛋白分子和细丝的研究远少于心脏，这将使我们能够剖析其中的关键。 IHM 相互作用限制两个头部的活动，关闭它们，从而在放松状态下保存 ATP。 使用单粒子 EM 和冷冻电镜来定义厚层中近原子水平 SRX 态的结构基础 兔子骨骼肌的细丝和肌球蛋白分子与狼蛛相比，显示出十倍- 更大的节能（超放松，HRX），我们将更深入地了解ATPase的机制 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 目标 2 将定义骨骼肌球蛋白头和重肌球蛋白中 SRX 的结构基础。 通过评估以下因素来评估分子：(A) SRX 是否由特定头部构象产生，以及 (B) IHM 是否与 SRX 状态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至关重要的作用，但它的结构基础以及与 IHM 和 与其他粗丝蛋白（MyBP-C、肌联蛋白）的关系仍然未知，我们的研究将揭示 IHM 结构。 骨骼粗丝和肌球蛋白分子，阐明其与 SRX 状态以及 MyBP-C 和 肌动蛋白，并为放松的分子基础和治疗药物的影响提供重要的见解。