Structural basis of the super-relaxed state in human cardiac muscle

肌肉中的肌球蛋白丝表现出一种节能的“超松弛”（SRX）状态， 是能量学和收缩调节的基础。在心肌（包括人类）中，SRX状态 有助于能源经济的螯合肌球蛋白头部的比例远离肌动蛋白， 当心脏活动增加时需要。SRX状态的病理改变被认为是许多 遗传性心肌病和治疗药物似乎通过逆转这些变化起作用。尽管 由于SRX的普遍性和重要性，其结构基础尚未定义，这在我们的研究中留下了一个关键的空白 了解心脏收缩和疾病的机制及其治疗。一个普遍的观点是， SRX在结构上与肌肉肌球蛋白的另一个普遍存在的特征相关：“相互作用头基序”（IHM）， 其中肌球蛋白的两个头部（封闭的和自由的）彼此相互作用并与近端肌球蛋白尾部（S2）相互作用， 抑制其活性并保存ATP。然而，最近的研究表明，SRX可能是一个属性， 肌球蛋白头部本身，而不需要头部相互作用。在本补助金中，我们将使用单粒子电子 显微镜（EM），冷冻EM和其他生物物理技术来定义SRX状态的结构基础， 关键肥厚型（HCM）和扩张型（DCM）心肌病诱导突变和治疗的影响 药物对IHM结构的影响 目的1将在分离的心肌肌球蛋白头（S1）和重肌球蛋白中确定SRX的基础 (HMM)通过评估是否：（A）SRX直接来自肌球蛋白头部的特定构象，以及（B） IHM与SRX状态相关。肌球蛋白构建体，其包含单头（S1）或两个头， 我们的合作者Dr。 克里斯托弗·延戈。对照组将有两个头，但只有2个七尾（2-hep），这不能形成完整的 IHM。负染色EM和类平均将揭示S1和IHM构象，冷冻EM将显示 第一次，近原子分辨率的结构揭示了IHM内部的相互作用， 放松.目标2将通过确定近-近-远 从心肌分离的细丝的原子冷冻电镜结构。目标3将揭示关键的结构性影响 HCM和DCM诱导的肌球蛋白头和IHM结构突变和治疗药物，使用冷冻EM 单粒子成像 SRX现在被广泛认为是正常放松肌肉的基本状态，但其结构 基础不理解。我们的高分辨率结构研究将揭示心脏的近原子结构 S1和IHM以及它们与分子和细丝中SRX的关系。这将提供新的见解 深入了解心肌松弛的基本机制， 心肌病的结构基础及其治疗。

Myosin filaments in muscle exhibit an energy-saving “super-relaxed” (SRX) state that is thought to be fundamental to the energetics and regulation of contraction. In cardiac muscle (including human), the SRX state contributes to energy economy by sequestering a proportion of myosin heads away from actin, to be released as needed when cardiac activity increases. Pathologic alterations to the SRX state are thought to underlie many inherited cardiomyopathies, and therapeutic drugs appear to work by reversing these changes. Despite its ubiquity and importance, the structural basis of the SRX has not been defined, leaving a crucial gap in our understanding of cardiac contraction and the mechanism of disease and its treatment. A widely held view is that SRX is structurally related to another ubiquitous feature of muscle myosin: the “interacting-heads motif” (IHM), in which myosin’s two heads (blocked and free) interact with each other and with the proximal myosin tail (S2), inhibiting their activity and conserving ATP. However, recent studies suggest that SRX may be a property of the myosin heads themselves, and not require head interactions. In this grant we will use single particle electron microscopy (EM), cryo-EM, and other biophysical techniques to define the structural basis of the SRX state and the impact of key hypertrophic (HCM) and dilated (DCM) cardiomyopathy-inducing mutations and therapeutic drugs on the structure of the IHM. Aim 1 will define the basis of SRX in the isolated cardiac myosin head (S1) and heavy meromyosin (HMM) by assessing if: (A) the SRX results directly from a specific conformation of the myosin heads, and (B) the IHM correlates with the SRX state. Myosin constructs comprising single heads (S1) or two heads with 15 heptads of tail (15-hep), enough to form the IHM, will be expressed and characterized by our collaborator, Dr. Christopher Yengo. Controls will have both heads but only 2 heptads of tail (2-hep), which cannot form a full IHM. Negative staining EM and class averaging will reveal S1 and IHM conformations, and cryo-EM will show for the first time the near-atomic resolution structure revealing the interactions within the IHM that underlie cardiac relaxation. Aim 2 will define the structural basis of the SRX in native thick filaments by determining the near- atomic cryo-EM structure of filaments isolated from cardiac muscle. Aim 3 will reveal the structural impact of key HCM- and DCM-inducing mutations and therapeutic drugs on myosin head and IHM structure, using cryo-EM and single particle imaging. The SRX is now widely recognized as a fundamental state of normal relaxed muscle, but its structural basis is not understood. Our high-resolution structural studies will reveal the near-atomic structures of cardiac S1 and the IHM and their relationship to the SRX in both molecules and filaments. This will provide new insights into the fundamental mechanism of relaxation (diastole) in cardiac muscle, and an improved understanding of the structural basis of cardiomyopathies and their treatment.