Structure and Mechanism of Cardiomyopathy Myosin Mutants

描述(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心肌病和扩张型心肌病的原因。该提案的目的是表征突变的人心肌肌球蛋白的生物物理和生化特性，作为鉴定导致遗传性心肌病的结构和机制变化的必要的第一步。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生产足够数量的高质量的人β-心肌肌球蛋白来进行结构和动力学分析。我们开发了一种基于C2C12细胞的腺病毒感染的哺乳动物表达系统，使我们能够产生大量的人β-心脏运动域和重肌球蛋白，这是动力学和结构研究所需的。野生型β-心脏马达结构域的晶体结构显示，在SH-1螺旋、转换结构域、楔形环和继电器螺旋/中继环(耦合区)通过一簇疏水残基(疏水NEXUS)连接的区域有一簇HCM和DCM突变。这些元素的协同运动对于肌球蛋白马达中的机械力-化学耦合至关重要。马达结构域与小分子激活剂omecamtiv meccarbil的对接和结构研究表明，它与疏水的Nexus结合在一起，可能通过影响机械力化学耦合机制发挥作用。本研究选择了8个HCM/DCM突变株，分别代表位于偶联区的良性和重度表型，用稳态和快速动力学方法研究突变马达的结构和机制的变化(目标1)。体外动力分析和单分子力测量(目标2.)和晶体结构(目标3)。美卡比利对突变的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启示。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.