Molecular Mechanism of Kinesin-2 Motility

描述（由申请人提供）：Kinesin-2马达在纤毛和鞭毛中进行顺行运输，以及细胞中的其他双向运输过程。虽然机械化学动力学的许多方面已经被理解，但在多电机和双向货物运输的基础上的具体运动活动和调节还没有被很好地理解。运动蛋白-2运输缺陷导致发育异常、光感受器降解和多囊肾病。这个项目的目标是了解Kinesin-2马达沿着微管行走的机制，以及Kinesin-2马达特性为其特定运输任务而调整的程度。与典型的Kinesin-1马达家族的一个显著区别是，Kinesin-2的颈部连接结构域（连接核心马达头部和卷曲线圈结构域）是17个氨基酸，而Kinesin-1只有14个氨基酸。由于颈部连接器作为机械元件将每个头部连接到它们的共享线圈，因此预计延长颈部连接器将减少头部区域之间的机械化学耦合。与此一致的是，我们发现延长Kinesin-1颈连接子会降低其加工能力，而缩短Kinesin-2颈连接子会提高加工能力。此外，我们发现Kinesin-2速度和运行长度的力依赖关系与Kinesin-1不同，这表明电机可能最适合双向货物运输，而不是长距离单向运输。通过单分子和多马达实验，结合kinesin化学-机械循环的计算建模，我们将揭示kinesin -1和kinesin -2马达之间机制差异的结构基础，目的是了解体内的双向运输。这项工作涉及结构研究，以确定颈连接器和颈线圈结构域在微管的进程运动中所起的作用。我们还将研究Kinesin-2化学机械循环中的具体步骤，以确定运动生物化学是如何由两个头结构域之间的分子内张力以及光学镊子施加的外部负载控制的。这些对Kinesin-2运动机制的研究将为理解Kinesin-2在细胞中的运输提供一个框架。通过对激酶1进行基准测试，这些测量将建立激酶机械化学的普遍主题，这将有助于更好地理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激酶的抗肿瘤疗法。

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. PUBLIC HEALTH RELEVANCE: Kinesin-2 motors transport intracellular cargo along microtubule filaments in the cell and in cilia and flagella, and deficiencies in Kinesin-2 transport lead to abnormal development, photoreceptor degradation and polycystic kidney disease. This study will investigate the molecular mechanism of Kinesin-2 motility to understand the cellular role of this motor and to develop general paradigms for understanding kinesin-driven transport. Experimental approaches include single-molecule studies and measuring biochemical kinetics, and results will be interpreted in the context of mathematical models of this mechanoenzyme.