Structural Framework for Understanding Myosin Thick-Filament Cardiomyopathies

描述(申请人提供)：健康肌肉的收缩不仅取决于功能上的分子马达，而且还取决于允许力量传递的健全的结构框架。在骨骼肌和心肌中，这种骨架是通过将肌动蛋白附着在相对的Z线上，以及主要由肌球蛋白的盘绕区域组装而成的双极粗丝提供的。粗大的细丝是一个紧密的集合，显示出肌球蛋白头部规则的螺旋排列。这意味着有一个潜在的结构性组织。肌球蛋白含有形成这种双极细丝所需的信息，然而，即使经过50多年的研究，这种粗大细丝的分子组织 目前仍不清楚。关于肌球蛋白杆在初级序列水平和组织粗丝的超微结构水平上都有丰富的知识。相比之下，关于肌球蛋白棒的高分辨率结构数据很少。因此，它 已经不可能为这种粗大的细丝生成分子模型。缺乏了解的原因是肌球蛋白棒的孤立片段形成了不适合高分辨率结构研究或分子表征的准晶体。这个问题现在已经通过加入适当的增溶结构域得到了解决，这使得可以通过X射线结晶学来确定人心肌肌球蛋白C末端区域的组装能力结构域的结构。肌球蛋白分子的这一部分对于双极纤维的形成是必不可少的。这项提议的目的是确定这种方法是否可以应用于产生整个肌球蛋白棒的高分辨率结构模型，以及该模型是否可以用于研究粗丝中肌球蛋白分子之间的相互作用。第一个具体目标是确定肌球蛋白杆段的高分辨率结构，这些结构已被证明影响组装，并确定导致双极细丝形成的分子特征。作为这项研究的一部分，将测量肌球蛋白棒远端节段之间的分子相互作用。与这些结构一起，这些生物物理测量将建立创建粗丝模型所必需的基本分子信息。目前，对于位于肌球蛋白棒的心脏和骨骼肌病突变的有害影响，尚无令人满意的生化解释。这是由于缺乏对粗丝的稳健模型。这项提议的第二个具体目的是利用这里确定的片段的结构来启动结构和生物物理研究，目的是在粗丝的计算模型的背景下为这些遗传损伤提供分子解释。从这项研究中获得的洞察力将适用于所有肌球蛋白II。因此，长期目标是扩展这里开发的方案，以研究肌球蛋白细丝的结构和组装。

DESCRIPTION (provided by applicant): Muscle contraction in healthy muscle depends not only on a functional molecular motor but also on a sound structural framework that allows for the transmission of force. In skeletal and cardiac muscle this framework is provided by the attachment of actin to the opposing Z-lines and by the bipolar thick filament assembled primarily from the coiled-coil region of myosin. The thick filament is a compact assembly that shows a regular helical disposition of myosin heads. This implies that there is an underlying structural organization. Myosin contains the information necessary to form this bipolar filament, however, even after more than fifty years of investigation, the molecular organization of the thick filament is still unclear. There is a wealth of knowledge concerning the myosin rod at the level of primary sequence and also at the ultrastructural level for the organization of the thick filament. In contrast, there is very little high resolution structural data for the myosin rod. Consequently, it has been impossible to generate a molecular model for the thick filament. The reason for the lack of knowledge is that isolated fragments of the myosin rod form paracrystals that are unsuitable for high resolution structural studies or molecular characterization. This problem has now been solved through the incorporation of appropriate solubilization domains, which has allowed the structure of the Assembly Competence Domain from the C-terminal region of human ??cardiac myosin to be determined by X- ray crystallography. This section of the myosin molecule is essential for bipolar filament formation. The purpose of this proposal is to determine whether this approach can be applied to yield a high resolution structural model for the entire myosin rod and whether this model can be utilized to investigate the interactions between myosin molecules in the thick filament. The first specific aim is to determine the high resolution structure for sections of the myosin rod that have been shown to influence assembly and to determine the molecular features responsible for the formation of bipolar filaments. As part of this study the molecular interactions between distal segments of the myosin rod will be measured. Together with the structures these biophysical measurements will establish the fundamental molecular information necessary to create a model for the thick filament. At present there is no satisfactory biochemical explanation for the deleterious effect of the cardiac and skeletal myopathy mutations located in the myosin rod. This is due to the lack of a robust model for the thick filament. The second specific aim of this proposal is to utilize the structure f fragments determined here to initiate structural and biophysical studies directed towards providing a molecular explanation for these genetic lesions within the context of a computational model for the thick filament. The insight gained from this study will be applicable to all myosin IIs. Thus, the long term goal is to extend the protocols developed here to investigate the structure and assembly of smooth muscle myosin filaments.