Structure and Mechanism of Cardiomyopathy Myosin Mutants

描述（由申请人提供）：β-心肌肌球蛋白重链突变是导致大量遗传性肥厚性（HCM）和扩张性心肌病（DCM）的原因。该提案的目的是表征突变的人类心肌肌球蛋白的生物物理和生化特性，作为确定导致遗传性心肌病的结构和机制变化的重要的第一步。研究人心肌肌球蛋白的困难是由于从患者组织中获得的蛋白质的不稳定性和异质性，以及缺乏足够的表达系统来产生高质量的人β-心肌肌球蛋白，其数量足以进行结构和动力学分析。我们开发了一种基于腺病毒感染C2C12细胞的哺乳动物表达系统，使我们能够生产动力学和结构研究所需的mg量的人β -心肌运动结构域和重肌球蛋白。野生型β-心脏运动结构域的晶体结构显示，在SH-1螺旋、转换结构域、楔形环和接力螺旋/接力环（耦合区）之间通过疏水残基（疏水Nexus）连接的区域存在HCM和DCM突变簇。这些元素的协同运动对于肌凝蛋白运动中的机械化学耦合至关重要。运动域与小分子激活剂omecamtiv mecarbil的对接和结构研究表明，它结合在疏水Nexus附近，可能通过影响机械化学偶联机制起作用。通过稳态和快速动力学（Aim 1.）、体外运动测定、单分子力测量（Aim 2.）和晶体结构（Aim 3 .）研究突变体运动结构和机制的变化，分别代表耦合区良性和严重表型的8个HCM/DCM突变体。奥米卡美对突变型心肌肌球蛋白偶联机制的影响可能为DCM患者的临床治疗提供新的见解。了解心肌病的分子机制将为开发新的靶向治疗方法提供基础。

DESCRIPTION (provided by applicant): Mutations in the β-cardiac myosin heavy chain are responsible for a large number of inherited Hypertrophic (HCM) and Dilated Cardiomyopathies (DCM). The objective of the proposal is the characterization of biophysical and biochemical properties of the mutant human cardiac myosin as an essential first step in identifying the changes in the structure and mechanism resulting in inherited cardiomyopathies. The difficulties in studying human cardiac myosin were due to the instability and heterogeneity of the protein obtained from patient tissues and the lack of an adequate expression system to produce high quality human β-cardiac myosin in quantities that are sufficient for structural and kinetic analysis. We developed a mammalian expression system based on adenoviral infection of C2C12 cells enabling us to produce mg quantities of the human β -cardiac motor domain and heavy meromyosin required for kinetic and structural studies. The crystal structure of the wild type β-cardiac motor domain reveals a cluster of HCM and DCM mutations in a region linking the SH-1 helix, the converter domain, the wedge loop, and the relay helix/relay loop (Coupling Region) by a cluster of hydrophobic residues (Hydrophobic Nexus). The concerted movement of these elements is critical for mechanochemical coupling in the myosin motor. Docking and structural studies of the motor domain with the small molecule activator, omecamtiv mecarbil, indicate that it binds adjacent to the Hydrophobic Nexus and may act by influencing the mechanochemical coupling mechanism. Eight HCM/DCM mutants representing the benign and severe phenotypes residing in the Coupling Region were selected to study the changes in the structure and mechanism of the mutant motor by steady-state and rapid kinetics (Aim 1.) in vitro motility assays and single molecule force measurements (Aim 2.) and crystallographic structure (Aim 3). The effect of omecamtiv mecarbil on the coupling mechanism of mutant cardiac myosins may provide insights into the clinical management of the disease specifically for DCM patients. Understanding the molecular mechanism of cardiomyopathies will provide the basis for the development of new assays for targeted therapies.