Understanding the Mechanism and Regulation of the Human Cytoplasmic Dynein Complex

 描述（由申请人提供）：真核细胞的复杂性需要细胞内的组织、协调和运动。为了克服这些挑战，细胞利用ATP驱动的分子马达，其沿沿着细胞骨架轨道单向运输细胞内组分。驱动蛋白和细胞质动力蛋白马达促进双向运输 通过分别向微管（MT）的正端和负端移动，可以将各种货物运输。虽然动力蛋白的运动机理模型已经有了详细的描述，但是动力蛋白运动的机制和调控机制还没有完全阐明。我们发现S.酿酒酵母动力蛋白通过其两个催化结构域的不协调步进而在MT上行走，并且其作用机制与驱动蛋白的协调的双手交替步进显著不同。令人惊讶的是，尽管最近的进展动力蛋白的结构表征，其强烈的方向性偏好移动到MT负端的分子起源仍然不清楚。近年来，人动力蛋白重组表达系统的建立，首次为深入研究其分子机制打开了大门。令人惊讶的是，人类动力蛋白只表现出短暂的占有性，产生的力明显低于S。这与人动力蛋白在细胞内长距离运输大的细胞内货物的能力不一致。新的研究表明，当人类动力蛋白与其辅因子动力蛋白和货物结合衔接子BicD 2形成2.5 MDa三元复合物（称为DDB）时，它的持续合成能力被激活。在我们的初步工作中，我们表明，dynactin和BicD 2也显着增强人类动力蛋白的力的产生，这表明DDB复合物是一个强大的电机和驱动蛋白的强大对手时，连接到同一货物。这项提案的目标是剖析活跃的人类动力蛋白复合物的机制，并确定动力蛋白和BicD 2如何调节动力蛋白在双向货物运输过程中与驱动蛋白-1竞争的能力。我们有三个具体目标。首先，使用蛋白质工程和单分子成像，我们将确定动力蛋白的机械组件，产生其负端定向运动。我们还将通过冷冻电子显微镜（cryoEM）解决“反向”结构的MT结合结构，以揭示动力蛋白方向性的结构基础。其次，我们将确定哪些部分（S）的电机是负责其自身抑制和dynactin和BicD 2如何调节机械化学循环，步进模式和力产生的人动力蛋白的特点。第三，我们将利用纯化的人驱动蛋白和DDB复合物在体外重建MT上的双向货物运输，并揭示这些马达之间的“拔河”机制和调节。我们的目标的成功将显着推进人类动力蛋白的基本机械化学的理解，并了解它如何实现细胞内货物的逆行运输。

 DESCRIPTION (provided by applicant): The complexity of eukaryotic cells requires intracellular organization, coordination, and locomotion. To overcome these challenges, cells utilize ATP-driven molecular motors, which transport intracellular components unidirectionally along cytoskeletal tracks. Kinesin and cytoplasmic dynein motors facilitate bidirectional transport of a variety of cargos by moving towards the plus- and minus-ends of microtubules (MTs), respectively. Detailed mechanistic models exist for kinesin, but the mechanism and regulation of dynein motility are still emerging. We found that S. cerevisiae dynein walks on a MT through uncoordinated stepping of its two catalytic domains and its mechanism of action differs significantly from the coordinated hand-over- hand stepping of kinesin. Surprisingly, despite recent advances in structural characterization of dynein, the molecular origin of its strong directional preference to move towards the MT minus-end remains unclear. Recently, a recombinant expression system was developed for human dynein, opening the doors for detailed studies of its molecular mechanism for the first time. Surprisingly, human dynein exhibited only short possessive runs and produces significantly lower forces than S. cerevisiae dynein in vitro, inconsistent with the ability of human dynein to transport large intracellular cargos over long distances inside cells. New work has revealed that processivity of human dynein is activated when it forms a 2.5 MDa ternary complex (referred to as DDB) with its cofactor dynactin and a cargo binding adaptor BicD2. In our preliminary work, we showed that dynactin and BicD2 also significantly enhance human dynein's force generation, suggesting that the DDB complex is a strong motor and a formidable opponent of kinesin when attached to the same cargo. The goal of this proposal is to dissect the mechanism of active human dynein complexes and determine how dynactin and BicD2 regulate dynein's ability to compete against kinesin-1 during bidirectional cargo transport. We have three specific aims. First, using protein engineering and single-molecule imaging, we will identify the mechanical components of dynein that give rise to its minus-end directed motility. We will also solve the MT-bound structure of "reverse directionality" constructs via cryo-electron microscopy (cryoEM) to reveal the structural basis of dynein directionality. Second, we will identify which part(s) of the motor is responsible for its autoinhibition and characterize how dynactin and BicD2 regulate the mechanochemical cycle, stepping pattern and force generation of human dynein. Third, we will reconstitute bidirectional cargo transport on MTs in vitro using purified human kinesin and DDB complexes and reveal the mechanism and regulation of "tug-of-war" between these motors. Success of our aims will significantly advance the understanding of the fundamental mechanochemistry of human dynein and learn how it achieves retrograde transport of intracellular cargos.