STRUCTURAL BASIS OF KINESIN MOTOR MOVEMENT

运动蛋白、动力蛋白和肌球蛋白构成了分子马达的三个超家族，它们通过将ATP水解产生的能量转化为机械功，沿着细胞骨架细丝产生力。运动蛋白超家族包含超过100种不同的运动蛋白，这些运动蛋白沿着微管轨道移动，并为细胞器运输和细胞分裂等细胞内运动过程提供动力。本实验室的长期目标是阐明激酶超家族成员在水解ATP和沿微管转运时所经历的构象变化。激酶超家族成员含有高度保守的催化结构域，具有ATP水解和微管结合活性。超家族的原型成员Kinesin有两个相同的运动域。它是一个过程马达，经历许多ATP水解循环而不与微管分离。尽管取得了很大的进展，但关于ATP水解循环中激酶蛋白的构象变化以及激酶蛋白加工的机制的信息有限。这一建议解决了这两个基本问题。两个马达结构域的结构和配置将在atp酶循环的不同点上使用冷冻电子显微镜和荧光偏振显微镜对荧光标记在马达结构域上的激酶分子进行表征。目前，最受欢迎的解释动力过程的假说是一种交替机制。我们将通过测试模型的结构预测来研究这一假设，例如两个运动域交替构象的存在。我们还将寻找导致运动蛋白易位的关键构象变化的直接结构证据。我们试图回答以下具体问题：1)ATP与运动蛋白的结合是否使两个运动结构域之一向前移动？2) ADP与激酶蛋白结合是否会导致有序的无序转变，从而使马达向前移动？3)当两个马达沿着微管运动时，它们的构象会发生变化吗？这些研究结果将有助于在分子水平上阐明驱动蛋白将ATP水解转化为机械功的结构基础。鉴于运动蛋白在许多细胞过程中的核心作用以及微管作为抗癌治疗靶点的重要性，这项工作将为正常细胞功能提供新的见解，并可能导致新的化疗靶点的确定。

Kinesin, dynein and myosin constitute the three superfamilies of molecular motors that generate force along cytoskeletal filaments by converting the energy from ATP hydrolysis into mechanical work. The kinesin superfamily contains more than 100 different motor proteins that move along microtubule tracks and power intracellular motile processes such as organelle transport and cell division. The long term goal of this laboratory is to elucidate the conformational changes that kinesin-superfamily members undergo as they hydrolyze ATP and translocate along microtubules. Kinesin-superfamily members contain a highly conserved catalytic domain that possesses the ATP hydrolytic and microtubule binding activities. Kinesin, the archetypal member of the superfamily, has two identical motor domains. It is a processive motor that undergoes many ATP hydrolysis cycles without dissociating from the microtubule. Despite much progress, there is limited information available on the conformational changes kinesin undergoes during ATP hydrolytic cycles and on the mechanism responsible for kinesin's processivity. This proposal addresses these two fundamental issues. The structure and configuration of the two motor domains will be characterized at different points in the ATPase cycle using cryo- electron microscopy and fluorescence polarization microscopy on kinesin molecules fluorescently labeled on the motor domain. Currently, the favored hypothesis to explain kinesin processivity is a hand over hand mechanism. We wilt investigate this hypothesis by testing structural predictions of the model such as the presence of alternating conformations for both motor domains. We will also look for direct structural evidence of the key conformational changes that are though to cause kinesin translocation. We seek to answer the following specific questions 1) Does ATP binding to kinesin moves forward one of the two motor domains? 2) Does ADP binding to kinesin cause an order disorder transition that could move the motor forward? 3) Do the two motors of kinesin alternate conformations as they walk along a microtubule? The results of these studies will contribute to elucidate at a molecular level the structural basis by which kinesins transduce ATP hydrolysis into mechanical work. Given the central role of kinesins in many cellular processes and the importance of microtubules as a target for anti-cancer therapy, this work will provide new insights into normal cellular function and may lead to the identification of new chemotherapeutic targets.