EM Studies of Dimeric Molecular Motors

描述(申请人提供)：在肌球蛋白超家族的马达蛋白中，肌球蛋白II是唯一的细丝形成类。肌球蛋白II是含有两条相同的重链和两对轻链的六角体，基本轻链(ELC)和调节轻链(RLC)，以及延伸到LMM细丝形成区域的长卷曲二聚结构域S2。被“调节”的肌球蛋白亚型要么依赖于RLC的磷酸化，例如节肢动物、平滑肌和非肌肉肌球蛋白，要么取决于钙离子结合，例如软体动物的横纹肌。基于肌球蛋白的调节通常依赖于两个肌球蛋白头部之间的分子内相互作用。这种相互作用在肌肉中可见，最近在节肢动物的粗丝中也可见，这表明头-头相互作用和构象变化是肌球蛋白II调节的一般特征。生物化学和模型研究已经确定了构象变化的一些特征，其中包括围绕S2α螺旋的扭转运动，这取决于S2的长度。这些扭转运动可能会影响其他肌球蛋白功能，包括与肌动蛋白细丝的双头相互作用和前进运动。这项资助申请建议研究肌球蛋白调节的结构和功能特征，这些特征与卷曲结构域的长度有很强的相关性。肌球蛋白V是一种细胞质肌球蛋白，也形成与肌球蛋白II相似的致密抑制构象，但在肌球蛋白头部和货物结合域之间存在相互作用。肌球蛋白II和肌球蛋白V都在被抑制的构象中与肌动蛋白结合，但亲和力非常不同。虽然肌球蛋白V被广泛认为是一种前进的运动，需要两个头部同时结合肌动蛋白，但两个头部同时结合平滑肌肌球蛋白是有争议的。肌球蛋白II和肌球蛋白V的抑制状态都有共同的潜在原理。这个项目寻求关于双头运动功能和调节的这些和其他方面的答案。最后，传统动蛋白中被抑制的构象还涉及货物结合域和动蛋白头部之间的相互作用。这种构象可能与被抑制的肌球蛋白构象有结构和功能上的相似性，也将被研究。

DESCRIPTION (provided by applicant): Among the myosin super-family of motor proteins, myosin II is the sole filament forming class. Myosin IIs are hexamers containing 2 identical heavy chains and 2 pairs of light chains, the essential light chain (ELC) and the regulatory light chain (RLC) and a long coiled-coil dimerization domain, S2, that extends into the LMM filament forming domain. Myosin isoforms that are "regulated" depend either on RLC phosphorylation, e.g., arthropod, smooth muscle and nonmuscle myosins, or Ca2+ binding, e.g., molluscan striated muscles. Myosin based regulation has in common a dependency on intramolecular interactions between the two myosin heads. This interaction was visualized in smooth muscle myosin and recently in arthropod thick filaments suggesting that a head-head interaction and a conformational change to a compact structure is a general feature of myosin II regulation. Biochemistry and modeling studies have identified a number of the features of the conformational change among which are torsional motions about the S2 alpha-helices that are dependent on the length of S2. These torsional motions could affect other myosin functions, including double headed interactions with the actin filament and processive motion. This grant application proposes to investigate structural and functional features of regulation in smooth muscle myosin that have a strong dependency on the length of the coiled-coil domain. Myosin V, a cytoplasmic myosin also forms a compact inhibited conformation with some similarity to that of myosin II, but with an interaction between the myosin heads and the cargo binding domain. Both myosin II and myosin V bind actin when in the inhibited conformation, but with very different affinities. While myosin V is widely identified as a processive motor, which requires simultaneous actin binding by both heads, simultaneous binding of smooth muscle myosin by both heads is controversial. The inhibited states of both myosin II and myosin V have common underlying principles. This project seeks answers to these and other aspects of two headed motor function and regulation. Finally, inhibited conformations in conventional kinesin also involve an interaction between the cargo binding domains and the kinesin heads. This conformation may have structural and functional similarities with inhibited conformations of myosin and will also be investigated.