Structure and mechanism of the dynein motor

描述(申请人提供)：动力蛋白最先被发现为纤毛和鞭毛的运动提供动力的微管马达。随后，在大多数真核细胞中，一种细胞质形式的动力蛋白被发现将大量的货物(膜细胞器、核、mRNAs、蛋白质、微管和病毒)移动到微管的负端。动力蛋白或其调节蛋白的突变与先天性缺陷(如内翻、无脑畸形)有关，调节动力蛋白的运输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。尽管它在细胞生物学和医学上很重要，但与另外两种主要的细胞骨架运动蛋白--激动素和肌球蛋白相比，人们对动力蛋白的运动性知之甚少。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，而且由于其尺寸非常大(&gt；300 kDa)，结晶一直难以实现。在这项资助申请中，我们建议获得酵母胞质动力蛋白运动域的第一个晶体结构。通过使马达在不同的核苷酸状态下结晶，我们还将寻求在其ATPase周期的不同阶段获得动力蛋白的“快照”。这些X射线结晶学研究将得到单分子运动学研究的补充，以测试动力蛋白如何产生运动性。基于我们的晶体结构，我们将设计新的重组动力蛋白，使荧光染料和其他生物物理探针能够放置在动力蛋白马达上的特定位置。使用这种探针，我们将使用单分子分析来测量依赖核苷酸的马达构象变化，并测试它们是否对运动性重要。我们还将讨论dynein的4ATP结合的作用，并确定dynein在执行一步时是使用一个还是多个ATP。最后，我们将开始对第二类细胞质动力蛋白的体外运动性研究，这些动力蛋白涉及从纤毛/鞭毛尖端向细胞体运输蛋白质。总之，这些研究将提供有关动力蛋白结构的新信息，它如何利用核苷酸并改变其构象，以及它如何适应细胞质和鞭毛中独特的运输功能。在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+ATPase超家族的一员，因此我们的研究结果将对理解这个ATPase大家族有价值。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想法。

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.