Structure and mechanism of the dynein motor

动力蛋白电机的结构和机理

• 批准号: 8518390
• 负责人: RONALD D VALE
• 金额: $ 22.09万
• 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/8518390
• 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）：Dynein首先被发现为基于微管的电动机，为Cilia和Flagella的运动提供动力。随后，发现动力蛋白的细胞质形式将大量的碳（膜细胞器，细胞核，mRNA，蛋白质，微管和病毒）移动到微管减去微管中的大多数真核细胞中。动力蛋白或其调节蛋白的突变与先天性缺陷有关（例如，现场倒置，lissencephaly），调节动力蛋白转运可能会为治疗病毒感染，癌症和神经退行性疾病提供新的策略。尽管它在细胞生物学和医学中的重要性，但与其他两个主要细胞骨架运动蛋白相比，基于动力蛋白的运动性知之甚少。动力蛋白场的主要缺陷是缺乏其运动域的原子分辨率结构信息，并且由于其非常大的尺寸（> 300 kDa）而难以实现结晶。在此赠款应用中，我们建议获得酵母细胞质动力蛋白的运动结构域的第一个晶体结构。通过在不同核苷酸状态下的电动机结晶，我们还将寻求在其ATPase循环的不同阶段获得动力蛋白的“快照”。这些X射线晶体学研究将通过单分子运动研究来补充，以测试动力蛋白如何产生运动。基于我们的晶体结构，我们将设计新的重组动力蛋白，以使荧光染料和其他生物物理探针放置在Dynein Motor上的定义位置。使用此类探针，我们将使用单分子测定法测量电动机的核苷酸构象变化，并测试它们是否对运动重要。我们还将解决Dynein的4 ATP结合的作用，并确定Dynein采取一步时是否使用一个或多个ATP。最后，我们将开始对第二类的细胞质动力蛋白进行的首次体外运动研究，该研究参与将蛋白质从纤毛/鞭毛的尖端传输到细胞体。总而言之，这些研究将提供有关动力蛋白结构，如何利用核苷酸和改变其构象的新信息，以及如何适应细胞质和鞭毛中独特的运输功能。这项工作中产生的试剂和结构将对整个Dynein领域具有广泛的价值。 Dynein是AAA+ ATPase超家族的成员，因此我们的研究结果对于理解这一大型ATPases家族非常有价值。我们的结构研究还将提供有关如何通过细胞调节蛋白以及可能通过治疗药物调节动力蛋白的新想法。