Molecular Mechanism of Kinesin-2 Motility

描述（由申请人提供）：驱动蛋白-2马达在纤毛和鞭毛中进行顺行运输，以及在细胞中进行其他双向运输过程。虽然许多方面的驱动蛋白机械化学的理解，具体的电机活动和调节，多电机和双向货物运输的基础还没有很好地理解。Kinesin-2转运缺陷导致发育异常、光感受器降解和多囊肾病。本项目的目标是了解驱动蛋白-2马达沿着微管行走的机制，以及驱动蛋白-2马达特性针对其特定运输任务进行调整的程度。与典型的驱动蛋白-1运动家族的显著差异是驱动蛋白-2的颈连接结构域（其将核心运动头连接到卷曲螺旋结构域）是17个氨基酸，而驱动蛋白-1中仅为14个氨基酸。因为颈连接体用作将每个头部连接到它们共享的螺旋-螺旋的机械元件，所以预期延伸颈连接体将减少头部结构域之间的机械化学偶联。与此一致，我们发现延长驱动蛋白-1颈连接子降低了其持续合成能力，缩短驱动蛋白-2颈连接子增强了持续合成能力。此外，我们发现驱动蛋白-2的速度和运行长度的力依赖性不同于驱动蛋白-1，这表明电机可能是最佳的双向货物运输，而不是长距离单向运输。使用单分子和多电机实验结合计算建模的驱动蛋白化学机械循环，我们将揭示驱动蛋白-1和驱动蛋白-2电机之间的机械差异的结构基础，了解体内双向运输的目标。这项工作涉及结构研究，以确定所发挥的作用，颈部连接器和颈部线圈结构域的进行性运动沿着微管。我们还将研究驱动蛋白-2化学机械循环中的具体步骤，以确定马达生物化学如何通过两个头部结构域之间的分子内张力以及通过光镊施加的外部负载来控制。这些对驱动蛋白-2运动机制的研究将为理解驱动蛋白-2在细胞中的转运提供一个框架。通过对驱动蛋白-1进行基准测试，这些测量将建立驱动蛋白机械化学的普遍主题，这将有助于更好地了解神经退行性疾病的分子基础，并有助于开发针对有丝分裂驱动蛋白的抗肿瘤疗法。

DESCRIPTION (provided by applicant): Kinesin-2 motors carry out anterograde transport in cilia and flagella, as well as other bidirectional transport processes in cells. Although many aspects of kinesin mechanochemistry are understood, the specific motor activities and regulation that underlie multi-motor and bidirectional cargo transport are not well understood. Deficiencies in Kinesin-2 transport lead to abnormal development, photoreceptor degradation and polycystic kidney disease. The goals of this project are to understand the mechanism by which Kinesin-2 motors walk along microtubules, and the degree to which Kinesin-2 motor properties are tuned for its specific transport tasks. A notable difference from the canonical Kinesin-1 motor family is that neck linker domain of Kinesin-2, which connects the core motor head to the coiled-coil domain, is 17 amino acids compared to only 14 in Kinesin-1. Because the neck linker serves as a mechanical element connecting each head to their shared coil-coil, it is expected that extending the neck linker will diminish mechanochemical coupling between the head domains. Consistent with this, we found that extending the Kinesin-1 neck linker diminishes its processivity and shortening the Kinesin-2 neck linker enhances processivity. In addition, we found that the force dependence of Kinesin-2 velocity and run length differ from Kinesin-1, suggesting the motor may be optimally tuned for bidirectional cargo transport rather than long distance unidirectional transport. Using single-molecule and multi-motor experiments in conjunction with computational modeling of the kinesin chemomechanical cycle, we will uncover the structural basis of mechanistic differences between Kinesin-1 and Kinesin-2 motors, with the goal of understanding bidirectional transport in vivo. This work involves structural studies to determine the role played by the neck linker and neck coil domains in processive movement along microtubules. We will also investigate specific steps in the Kinesin-2 chemomechanical cycle to determine how the motor biochemistry is controlled by intramolecular tension between the two head domains, as well as by external loads applied by optical tweezers. These investigations into the mechanism of Kinesin-2 motility will provide a framework in which to understand Kinesin-2 transport in cells. By benchmarking against Kinesin-1, these measurements will establish universal themes underlying kinesin mechanochemistry that will help to better understand the molecular basis of neurodegenerative diseases and aid the effort to develop anti-tumor therapies targeting mitotic kinesins.