Structure and mechanism of the dynein motor

描述（由申请人提供）：动力蛋白首次被发现是基于微管的马达，为纤毛和鞭毛的运动提供动力。随后，在大多数真核细胞中，细胞质形式的动力蛋白被发现将许多货物（膜细胞器、细胞核、mRNA、蛋白质、微管和病毒）向微管负端移动。动力蛋白或其调节蛋白的突变与先天性缺陷（例如，原位反位，无脑畸形）相关，调节动力蛋白转运可能为治疗病毒感染，癌症和神经退行性疾病提供新的策略。尽管其在细胞生物学和医学中的重要性，基于动力蛋白的运动性与驱动蛋白和肌球蛋白（另外两种主要的细胞骨架运动蛋白）相比知之甚少。动力蛋白领域的一个主要缺陷是缺乏其马达结构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。在这个授权申请中，我们建议获得酵母细胞质动力蛋白的马达结构域的第一个晶体结构。通过在不同核苷酸状态下结晶马达，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射线晶体学研究将补充单分子运动研究，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，这将使荧光染料和其他生物物理探针在动力蛋白马达上的定义位置的位置。使用这样的探针，我们将通过单分子测定来测量马达的核苷酸依赖性构象变化，并测试它们是否对运动性重要。我们还将讨论动力蛋白的4 ATP结合的作用，并确定动力蛋白是否使用一个或多个ATP时，它采取了一个步骤。最后，我们将着手进行第二类细胞质动力蛋白的第一次体外运动研究，这些动力蛋白参与将蛋白质从纤毛/鞭毛的尖端运输到细胞体。总之，这些研究将为动力蛋白的结构，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提供新的信息。在这项工作中产生的试剂和结构将是广泛有价值的整个动力蛋白领域。动力蛋白是AAA+ ATP酶超家族的一员，因此我们的研究结果对于理解这个ATP酶大家族将是有价值的。我们的结构研究也将为动力蛋白如何被细胞调节蛋白以及潜在的治疗药物调节提供新的思路。

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.