Structural basis of motility by dimeric kinesin motor proteins

描述（由申请人提供）：驱动蛋白分子马达通过与一对催化头部结构域采取交替步骤沿着沿着微管移动，其中每个步骤由单个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的分子细节是相当有意义的，不仅作为理解这种马达的活性如何在体内被各种调节因子调节的基础，而且还有助于开发针对这些马达用于癌症治疗和其他治疗目的的药物。尽管深入的研究，然而，驱动蛋白的运动周期的基础构象变化仍然存在很大的争议。一个特别难以理解的问题是二聚体驱动蛋白如何维持连续的步进运动，因为现有的方法还没有捕获主动步进驱动蛋白二聚体的结构。最近，我们在驱动蛋白马达的研究中取得了两项突破。首先，通过使用最先进的冷冻电子显微镜仪器与我们自己的新的图像处理方法相结合，我们已经解决了驱动蛋白微管复合物的新的3D重建，分辨率约为5-6？，大大提高了以前的努力。这张图揭示了微管刺激ADP释放后驱动蛋白活性位点的意外重排，这表明驱动蛋白循环中这一关键步骤的新机制，也为电机的动力冲程提供了信息。第二，我们已经设计了一种新的算法，用于产生高分辨率的三维重建从冷冻EM图像的不完美的装饰，异质组件的驱动蛋白与微管。这种方法使我们能够解决第一个三维重建的驱动蛋白二聚体，因为它的步骤沿着微管。我们将联合收割机结合我们的新的冷冻电镜方法与其他国家的最先进的结构和功能技术，包括原子力显微镜和饱和转移EPR主机，建立驱动蛋白运动功能的详细基础。通过比较结构和功能特性的二聚体驱动蛋白的存在或不存在的突变，导致运动协调的损失，我们将定义的结构基础的分子间的张力控制和其他关键特性的驱动蛋白，使二聚化。我们还将应用cryo-EM对驱动蛋白催化结构域中的定点突变体进行结构/功能研究，以检验驱动蛋白活性如何受以下因素调节的假设： 微管结合，以及马达如何在其周期中调节其对微管的亲和力。 在这项研究过程中开发的方法将改变我们研究许多其他大型和以前难以处理的免疫结合蛋白的能力，包括其他分子马达家族以及微管切断酶。

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.