Structural basis of motility by dimeric kinesin motor proteins

描述（由申请人提供）：Kinesin分子马达通过一对催化头结构域的交替步骤沿着微管移动，其中每一步都由单个ATP分子的水解提供动力。这种活性在许多细胞功能中起关键作用，如有丝分裂和神经元囊泡运输。因此，解剖驱动蛋白运动功能背后的分子细节是相当有趣的，这不仅是理解这种运动的活动如何在体内被各种调节因子调节的基础，而且有助于开发针对这些运动的药物，用于癌症治疗和其他治疗目的。然而，尽管有深入的研究，驱动蛋白运动周期的构象变化仍然存在激烈的争论。一个特别难以捉摸的问题是二聚体驱动蛋白是如何维持连续的阶梯式运动的，因为现有的方法还没有捕捉到主动步进驱动蛋白二聚体的结构。我们最近在马达的研究上取得了两项突破。首先，通过结合使用最先进的低温电子显微镜仪器和我们自己的新颖图像处理方法，我们以~5-6¿分辨率解决了新的动力微管复合物的3D重建，大大改善了以前的努力。这张图揭示了微管刺激ADP释放后，肌动蛋白活性位点的意外重排，提示了肌动蛋白周期中这一关键步骤的新机制，也通知了马达的功率行程。其次，我们设计了一种新的算法，用于从冷冻电镜图像中产生高分辨率的3D重建，这些图像是带有微管的不完美修饰的异质驱动蛋白组装。这种方法使我们能够解决第一个三维重建的运动蛋白二聚体，因为它沿着微管的步骤。我们将结合我们新的冷冻电镜方法与许多其他最先进的结构和功能技术，包括AFM和饱和转移EPR，以建立运动功能的详细基础。通过比较二聚体驱动蛋白在存在或不存在导致运动协调丧失的突变的情况下的结构和功能特性，我们将定义分子间张力控制的结构基础以及二聚化激活的驱动蛋白的其他关键特性。我们也将冷冻电镜应用于激酶催化区域的位点导向突变体的结构/功能研究，以测试激酶活性如何被调节的假设

DESCRIPTION (provided by applicant): Kinesin molecular motors move along microtubules by taking alternating steps with a pair of catalytic head domains, where each step is powered by hydrolysis of a single molecule of ATP. This activity plays a key role in numerous cellular functions such as mitosis and neuronal vesicle transport. It is therefore of considerable interest to dissect the molecular details that underlie kinesin's motility functions, not only as a basis fo understanding how this motor's activity may be modulated in vivo by a large variety of regulating factors, but also to aid the development of pharmaceuticals that target these motors for cancer therapy and other therapeutic purposes. Despite intensive study, however, the conformational changes that underlie kinesin's motility cycle remain strongly debated. A particularly elusive question is how dimeric kinesin sustains continuous stepwise movement, because existing methods have not captured the structure of actively stepping kinesin dimers . We have recently made two breakthroughs in our studies of the kinesin motor. First, by using a combination of state of the art cryo-electron microscopy instrumentation together with our own novel image-processing methods, we have solved a new 3D reconstruction of the kinesin-microtubule complex at ~5-6¿ resolution, substantially improving on previous efforts. This map reveals an unanticipated rearrangement of kinesin's active site following microtubule-stimulated ADP release, suggesting a novel mechanism for this key step in the kinesin cycle and also informing the motor's power stroke. Second, we have devised a novel algorithm for producing high-resolution 3D reconstructions from cryo-EM images of imperfectly decorated, heterogeneous assemblies of kinesin with microtubules. This method has allowed us to solve the first 3D reconstruction of a kinesin dimer as it steps along a microtubule. We will combine our new cryo-EM methods with a host of other state of the art structural and functional techniques, including AFM and saturation-transfer EPR, to establish the detailed basis of kinesin motor function. By comparing structure and functional properties of dimeric kinesin in the presence or absence of mutations that cause loss of motor coordination, we will define the structural basis of inter-molecular tension control and other critical properties of kinesin that are enabled by dimerization. We will also apply cryo-EM to structure/function studies of site-directed mutants in the kinesin catalytic domain in order to test hypotheses for how kinesin's activity is regulated by microtubule binding, and how the motor regulates its affinity for the microtubule during its cycle. The methods developed during the course of this research will transform our ability to study many other large and previously intractable filament-binding proteins, including other molecular motor families as well as microtubule severing enzymes.