Structure and mechanism of the dynein motor

 描述(由申请人提供)：动力蛋白、动蛋白和肌球蛋白，三类细胞骨架马达蛋白，为真核细胞的大部分运动提供动力。在人类健康方面，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。控制肌球蛋白和运动蛋白马达活动的小分子药物(心力衰竭时上调心脏肌球蛋白活性或癌症时下调有丝分裂运动蛋白活性)目前正在进行临床试验。在三种类型的细胞骨架马达蛋白中，动力蛋白最不为人所知。虽然Kinesin和Dynein都是基于微管的马达蛋白，但它们有不同的结构和进化历史；Kinesin起源于G蛋白谱系，而更大的Dynein马达则是从AAA ATPase进化而来。这笔赠款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、mRNAs、染色体和病毒)绝大多数负端定向微管运动的马达。我们过去的资助主要集中在动力蛋白的X射线结晶学上。虽然我们将继续使用这种方法，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原子分辨率的结构。我们正在与加州大学旧金山分校的一名研究人员合作，他是这种方法的先驱。利用这些工具，我们提出了解决动力蛋白“前PowerStroke”状态的结构，补充了“PowerStroke后”状态的早期X射线结构。这些工作将完善我们对动力蛋白化学机械循环的看法，使我们能够理解ATPase周期中的转变如何触发跨大动力蛋白运动域的变构变化，从而产生运动性。为了补充这些结构快照，我们将使用单分子技术对活跃的循环动力蛋白马达的结构变化进行动态测量。我们还将使用稳定前的核苷酸结合测量来剖析动力蛋白的三个活性ATPase位点的作用。Kinesin和Myosin只有一个ATPase位点，所以我们希望解开Dynein如何利用它另外两个ATPase位点的谜团。上述工作将利用酵母动力蛋白运动区进行，该结构域具有结构性活性，是理解动力蛋白运动性的良好模型系统。然而，我们最近发现，哺乳动物细胞质动力蛋白更加复杂，需要一个货物适配器蛋白和动力蛋白(一种多亚单位蛋白复合体)才能完全激活。为了了解这一调控机制的结构基础，我们将获得大型动力蛋白-动力蛋白-适配器复合体的冷冻EM结构，以便了解这些组件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，在这一授权期结束时，我们希望为驱动动力蛋白运动的结构变化得出一个详细的模型，并阐明在哺乳动物细胞中调节动力蛋白的一个仍然鲜为人知的机制。

 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.