Structure and mechanism of the dynein motor

动力蛋白电机的结构和机理

• 批准号: 8643796
• 负责人: RONALD D VALE
• 金额: $ 22.87万
• 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 2015-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8643796
• 关键词: ATP phosphohydrolase; Address; Applications Grants; Binding; Biological Assay; Brain; Carrier Proteins; Cell Nucleus; Cell physiology; Cells; Cellular biology; Cilia; Complement; Congenital Abnormality; Congenital Heart Defects; Crystallization; Cytoplasm; Development; Dynein ATPase; Enzymes; Eukaryotic Cell; Family; Flagella; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Goals; Grant; Heavy Metals; In Vitro; Intracellular Transport; Kidney; Kinesin; Lead; Location; Malignant Neoplasms; Maps; Measures; Medicine; Membrane; Membrane Proteins; Messenger RNA; Microtubule Proteins; Microtubules; Mind; Minus End of the Microtubule; Mitotic spindle; Modeling; Molecular Conformation; Motion; Motor; Movement; Mutation; Myosin ATPase; Neurodegenerative Disorders; Nucleotides; Organelles; Pharmaceutical Preparations; Phase; Positioning Attribute; Property; Proteins; Reagent; Recombinants; Recording of previous events; Regulation; Relative (related person); Resolution; Roentgen Rays; Role; Side; Site; Situs Inversus; Solutions; Staging; Stroke; Structural Models; Structure; System; Testing; Therapeutic; United States National Institutes of Health; Virus; Virus Diseases; Work; Workplace; X-Ray Crystallography; Yeasts; base; cell motility; design; electron density; genetic regulatory protein; lissencephaly; member; neuronal cell body; polypeptide; post stroke; public health relevance; single molecule; small molecule; sperm cell

DESCRIPTION (provided by applicant): Dynein was first discovered as the microtubule-based motor that powers the movement of cilia and flagella. Subsequently, a cytoplasmic form of dynein was found to move numerous cargos (membrane organelles, the nucleus, mRNAs, proteins, microtubules, and viruses) towards the microtubule minus end in most eukaryotic cells. Mutations in dynein or its regulatory proteins have been associated with congenital defects (e.g. situs inversus, lissencephaly), and modulating dynein transport may provide new strategies for treating viral infections, cancer, and neurodegenerative disease. Despite its importance in cell biology and medicine, dynein-based motility is poorly understood in comparison to kinesin and myosin, the two other major cytoskeletal motor proteins. A major deficiency in the dynein field is the lack of atomic resolution structural information of its motor domain, and crystallization has been difficult to achieve because its very large size (>300 kDa). In this grant application, we propose to obtain the first crystal structure of the motor domain of yeast cytoplasmic dynein. By crystallizing the motor in different nucleotide states, we also will seek to obtain "snap shots" of dynein in different stages of its ATPase cycle. These X-ray crystallography studies will be complemented by single molecule motility studies to test how dynein produces motility. Based upon our crystal structure, we will design new recombinant dyneins that will enable placement of fluorescent dyes and other biophysical probes in defined locations on the dynein motor. Using such probes, we will measure by nucleotide-dependent conformational changes of the motor using single molecule assays and test whether they are important for motility. We also will address the role of dynein's 4 ATP binding and determine whether dynein uses one or multiple ATPs when it takes a step. Finally, we will embark on the first in vitro motility studies of a second class of cytoplasmic dyneins involved in transporting proteins from the tips of cilia/flagella to the cell body. In summary, these studies will provide new information on dynein's structure, how it utilizes nucleotides and changes its conformation, and how it has adapted for unique transport functions in the cytoplasm and the flagellum. The reagents and structures generated in this work will be broadly valuable to the entire dynein field. Dynein is a member of the AAA+ ATPase superfamily, and thus results of our studies will be valuable for understanding this large family of ATPases. Our structural studies also will provide new ideas on how dynein can be regulated by cellular regulatory proteins as well as potentially by therapeutic drugs.

描述（由申请人提供）：动力蛋白最初被发现是一种基于微管的马达，为纤毛和鞭毛的运动提供动力。随后，人们发现细胞质形式的动力蛋白可以将大量的货物（膜细胞器、细胞核、mRNA、蛋白质、微管和病毒）移向大多数真核细胞的微管负端。动力蛋白或其调节蛋白的突变与先天性缺陷（例如逆位、无脑畸形）有关，调节动力蛋白运输可能为治疗病毒感染、癌症和神经退行性疾病提供新策略。尽管动力蛋白在细胞生物学和医学中很重要，但与另外两种主要的细胞骨架运动蛋白驱动蛋白和肌球蛋白相比，人们对基于动力蛋白的运动知之甚少。动力蛋白领域的一个主要缺陷是缺乏其运动域的原子分辨率结构信息，并且由于其尺寸非常大（>300 kDa）而难以实现结晶。在这项拨款申请中，我们计划获得酵母细胞质动力蛋白运动结构域的第一个晶体结构。通过使马达结晶在不同的核苷酸状态，我们还将寻求获得动力蛋白在其 ATP 酶循环的不同阶段的“快照”。这些 X 射线晶体学研究将得到单分子运动研究的补充，以测试动力蛋白如何产生运动。根据我们的晶体结构，我们将设计新的重组动力蛋白，从而能够将荧光染料和其他生物物理探针放置在动力蛋白电机上的指定位置。使用此类探针，我们将使用单分子测定来测量马达的核苷酸依赖性构象变化，并测试它们对于运动是否重要。我们还将探讨动力蛋白 4 ATP 结合的作用，并确定动力蛋白在迈出一步时是否使用一个或多个 ATP。最后，我们将着手对第二类细胞质动力蛋白进行首次体外运动研究，该类细胞质动力蛋白参与将蛋白质从纤毛/鞭毛尖端转运到细胞体。总之，这些研究将提供关于动力蛋白结构、它如何利用核苷酸和改变其构象，以及它如何适应细胞质和鞭毛中独特的运输功能的新信息。这项工作中产生的试剂和结构将对整个动力蛋白领域具有广泛的价值。动力蛋白是 AAA+ ATP 酶超家族的成员，因此我们的研究结果对于了解这一大家族的 ATP 酶非常有价值。我们的结构研究还将提供关于如何通过细胞调节蛋白以及潜在的治疗药物调节动力蛋白的新思路。