Structure and mechanism of the dynein motor

 描述（由申请人提供）：动力蛋白、驱动蛋白和肌球蛋白是三类细胞骨架运动蛋白，为真核细胞的大部分运动提供动力。就人类健康而言，许多心脏、肾脏、听觉和神经系统疾病都与细胞骨架马达的突变有关。操纵肌球蛋白和驱动蛋白马达活性的小分子药物（上调心力衰竭的心肌肌球蛋白活性或下调癌症的有丝分裂驱动蛋白活性）目前正在临床试验中进行测试。在三种类型的细胞骨架运动蛋白中，动力蛋白的研究最少。虽然驱动蛋白和动力蛋白都是基于微管的马达蛋白，但它们具有不同的结构和进化历史;驱动蛋白出现于G蛋白谱系，而更大的动力蛋白马达从AAA ATP酶进化而来。这项资助的目标是了解细胞质动力蛋白运动的结构基础，细胞质动力蛋白是驱动细胞内货物（如膜，mRNA，染色体和病毒）的绝大多数负末端导向微管运动的马达。我们过去的资助主要集中在动力蛋白的X射线晶体学上。虽然我们将继续利用这种方法，但我们正在更多地转向电子显微镜，因为低温EM的最新进展可以产生具有原子分辨率的结构。我们正在与加州大学旧金山分校的一位研究人员合作，他是这种方法的先驱。有了这些工具，我们建议解决结构动力蛋白在其“前powerstroke”的状态，补充早期的X射线结构的“后powerstroke”的状态。这些工作将完成我们对动力蛋白化学机械循环的看法，使我们能够理解ATP酶循环中的转换如何触发产生运动性的大型动力蛋白运动域的变构变化。为了补充这些结构的“快照”，我们将使用单分子技术动态测量主动循环动力蛋白马达的结构变化。我们也将使用前稳态核苷酸结合测量来剖析动力蛋白的三个活性ATP酶位点的作用。驱动蛋白和肌球蛋白只有一个ATP酶位点，因此我们希望解决动力蛋白如何利用其两个额外的ATP酶位点的奥秘。上述工作将与酵母动力蛋白运动域，这是组成型活性和理解动力蛋白运动的一个很好的模型系统进行。然而，我们最近发现，哺乳动物细胞质动力蛋白更复杂，需要一个货物衔接蛋白和动力蛋白（多亚基蛋白复合物）变得完全活跃。为了了解这种调节机制的结构基础，我们将获得大型动力蛋白-动力蛋白-适配器复合物的冷冻EM结构，以了解这些组分如何相互作用以及这些相互作用如何导致动力蛋白激活。因此，在本资助期结束时，我们希望能够推导出驱动动力蛋白运动的结构变化的详细模型，并阐明哺乳动物细胞中调节动力蛋白的机制。

 DESCRIPTION (provided by applicant): Dynein, kinesin, and myosin, three classes of cytoskeletal motor proteins, power the majority of movements of eukaryotic cells. With regard to human health, many cardiac, kidney, auditory, and nervous system diseases have been linked to mutations in cytoskeletal motors. Small molecule drugs that manipulate the activities of myosin and kinesin motors (up-regulating cardiac myosin activity for heart failure or down-regulating mitotic kinesin activity for cancer) are now being tested in clinical trials. Of the thre types of cytoskeletal motor proteins, dynein is least well understood. While kinesin and dynein are both microtubule-based motor proteins, they have distinct structures and evolutionary histories; kinesin emerged from the G protein lineage, while the much larger dynein motor evolved from the AAA ATPases. The goal for this grant is to understand the structural basis of motility by cytoplasmic dynein, the motor that drives the vast majority of minus-end-directed microtubule motility of intracellular cargoes such as membranes, mRNAs, chromosomes and viruses. Our past grant focused on X-ray crystallography of dynein. While we will continue to utilize this approach, we are shifting more towards electron microscopy because of recent advances in cryo EM that can produce structures with atomic resolution. We are collaborating with an investigator at UCSF who is pioneering such approaches. With these tools, we propose to solve structures for dynein in its "pre-powerstroke" states, complementing earlier X-ray structures of the "post-powerstroke" states. Such work will complete our view of dynein's chemomechanical cycle, allowing us to understand how transitions in the ATPase cycle trigger allosteric changes across the large dynein motor domain which produces motility. To complement these structural "snap shots", we will make dynamic measurements of the structural changes in active, cycling dynein motors using single molecule techniques. We also will dissect the roles of dynein's three active ATPase sites using pre- steady state nucleotide binding measurements. Kinesin and myosin only have a single ATPase site, so we hope to resolve the mystery of how dynein utilizes its two additional ATPase sites. The above work will be performed with the yeast dynein motor domain, which is constitutively active and a good model system for understanding dynein motility. However, we recently discovered that mammalian cytoplasmic dynein is more complicated and requires a cargo adapter protein and dynactin (a multi-subunit protein complex) to become fully active. To understand the structural basis of this regulatory mechanism, we will obtain a cryo EM structure of the large dynein-dynactin-adapter complex in order to understand how these components interact and how these interactions lead to dynein activation. Thus, by the end of this grant period, we hope to derive a detailed model for the structural changes that drive dynein motility and illuminate a still poorly understood mechanism for regulating dynein in mammalian cells.