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

肌肉中的肌球蛋白细丝表现出一种节能的“超级放松”(SRX)状态，这种状态被认为是 是收缩的能量学和调节的基础。在心肌(包括人)中，SRX状态 通过将一部分肌球蛋白头部从肌动蛋白中隔离出来，从而有助于节约能源 当心脏活动增加时，根据需要进行调整。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。尽管它的 无处不在和重要，SRX的结构基础尚未定义，在我们的 了解心脏收缩和疾病的发病机制及其治疗。一种普遍的观点是 SRX在结构上与肌肉肌球蛋白的另一个普遍特征有关：“相互作用的头部基序”(IHM)， 其中肌球蛋白的两个头部(被阻挡的和自由的)相互作用并与近端的肌球蛋白尾巴(S2)相互作用， 抑制它们的活性，保存ATP。然而，最近的研究表明，SRX可能是 肌球蛋白头部本身，不需要头部相互作用。在这项研究中，我们将使用单粒子电子 显微镜(EM)、低温EM和其他生物物理技术，以定义SRX状态的结构基础和 关键肥厚型(HCM)和扩张型(DCM)心肌病诱发突变的影响及治疗 药物对IHM结构的影响。 目标1将确定SRX在分离的心肌肌球蛋白头部(S1)和重肌球蛋白中的基础 (HMM)通过评估：(A)SRX是否直接由肌球蛋白头部的特定构象产生，以及(B) IHM与SRX状态相关。肌球蛋白结构由单头(S1)或双头(15 七个尾巴(15-hep)，足以形成IHM，将由我们的合作者Dr。 克里斯托弗·延戈。控件将有两个头部，但只有两个七头尾巴(2-hep)，这不能形成完整的 嗯。负染色EM和分类平均将显示S1和IHM构象，而冷冻-EM将显示 近原子分辨率结构首次揭示了构成心脏的IHM内部的相互作用 放松一下。目标2将确定天然粗丝中SRX的结构基础，方法是确定近 从心肌中分离出的细丝的原子冷冻-EM结构。目标3将揭示KEY的结构影响 用冷冻-EM技术研究肌球蛋白头部和IHM结构中HCM和DCM诱导的突变和治疗药物 和单粒子成像。 SRX现在被广泛认为是正常放松肌肉的基本状态，但其结构性 基础是不理解的。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 探讨心肌松弛(舒张期)的基本机制，并加深对 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.