Structure and mechanism of the dynein motor

动力蛋白电机的结构和机理

• 批准号: 8886887
• 负责人: RONALD D VALE
• 金额: $ 27.72万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8886887
• 关键词: ATP phosphohydrolase; Address; Adenylyl Imidodiphosphate; Auditory; Binding; Binding Sites; Biological; Biological Models; Cardiac; Cardiac Myosins; Cell division; Cellular biology; Chemicals; Chromosome Segregation; Chromosomes; Cilia; Clinical Trials; Communities; Complement; Complex; Computer Analysis; Crystallization; Dictyostelium; Dynein ATPase; Electron Microscopy; Eukaryotic Cell; Family; Fluorescence Resonance Energy Transfer; Future; GTP-Binding Proteins; Goals; Grant; Health; Heart failure; Heterogeneity; Human; Intracellular Transport; Kidney; Kinesin; Kinetics; Laboratories; Lead; Link; Malignant Neoplasms; Mammalian Cell; Measurement; Measures; Membrane; Membrane Proteins; Membrane Transport Proteins; Messenger RNA; Methods; Microtubules; Mitotic; Mitotic Chromosome; Mitotic spindle; Molecular; Molecular Conformation; Molecular Motors; Monitor; Motor; Movement; Mutation; Myocardium; Myopathy; Myosin ATPase; Nerve Fibers; Nuclear; Nucleotides; Pharmaceutical Preparations; Play; Positioning Attribute; Power stroke; Property; Protein Complex Subunit; Proteins; RNA; Reaction; Recording of previous events; Research Personnel; Resolution; Roentgen Rays; Role; Rotation; Site; Skeletal Muscle; Structural Models; Structure; Techniques; Testing; Time; Trachea; United States National Institutes of Health; Vanadates; Virus; Vision; Work; X-Ray Crystallography; Yeasts; adapter protein; arm; base; cell motility; drug development; dynactin; fascinate; genetic regulatory protein; millisecond; nervous system disorder; particle; public health relevance; single molecule; small molecule; sperm cell; tool

 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.

 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之相关。细胞骨架马达的突变，可操纵肌球蛋白和驱动蛋白马达的活性（上调心肌肌球蛋白活性以治疗心力衰竭或下调）目前正在临床试验中测试三种类型的细胞骨架运动蛋白，其中动力蛋白是最不为人所知的，虽然驱动蛋白和动力蛋白都是基于微管的运动蛋白，但它们具有不同的结构和进化历史。来自 G 蛋白谱系，而更大的动力蛋白马达则由 AAA ATP 酶进化而来。这项资助的目标是通过以下方式了解运动的结构基础。细胞质动力蛋白，驱动细胞内物质（如膜、mRNA、染色体和病毒）绝大多数负端定向微管运动的发动机，我们过去的资助主要集中在动力蛋白的 X 射线晶体学上，而我们将继续利用它。由于冷冻电镜技术的最新进展，我们正在与加州大学旧金山分校的一位研究人员合作，他是此类方法的先驱。利用这些工具，我们建议解决动力蛋白“动力冲程前”状态的结构，补充早期“动力冲程后”状态的 X 射线结构，这样的工作将完善我们对动力冲程化学机械循环的看法，使我们能够理解动力冲程。 ATP酶循环中的转变如何触发产生运动的大动力蛋白运动域的变构变化为了补充这些结构“快照”，我们将对活跃循环中的结构变化进行动态测量。我们还将使用稳态前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，因此我们希望解开动力蛋白如何利用其的谜团。上述工作将使用酵母动力蛋白运动结构域进行，该结构域具有组成型活性，是理解动力蛋白的良好模型系统。然而，我们最近发现哺乳动物细胞质动力蛋白更加复杂，需要货物接头蛋白和动力蛋白（多亚基蛋白复合物）才能完全活跃。为了了解这种调节机制的结构基础，我们将获得冷冻。大型动力蛋白-动力蛋白-适配器复合物的 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，在本资助期结束时，我们希望推导出来。驱动动力蛋白运动的结构变化的详细模型，并阐明了哺乳动物细胞中仍知之甚少的调节动力蛋白的机制。