Computational and single molecule analysis of kinesin's atomistic machinery

描述（由申请人提供）：肌动蛋白是已知最小的两足运动蛋白，它使用ATP作为燃料沿着微管路径行进。它的正常功能对细胞内货物运输和细胞分裂等许多重要任务至关重要。因此，深入了解运动蛋白的功能不仅对推进分子马达的基础知识很重要，而且对开发针对涉及细胞内运输受损的疾病的新疗法也至关重要。虽然过去的进展揭示了其整体运动特征的许多重要方面，但其步进运动的物理机制仍不清楚。一般来说，研究运动蛋白或运动蛋白的一个主要困难是分子动态地感知并产生运动力，这很难仅仅基于静态结构图来考虑。为了研究原子细节的动力学方面，我们采用了分子动力学模拟和单分子实验的协同方法。通过分子动力学模拟，我们发现驱动蛋白通过一个结构域的折叠产生力，我们将其命名为“盖颈束”。虽然我们的单分子运动实验支持了所提出的机制，这些实验测试了旨在产生较少力的激酶突变体，但这些实验导致了关于力产生的能量学以及力产生步骤在整个激酶机械化学循环中的作用的进一步问题。此外，我们的初步模拟确定了运动蛋白运动的另外两个关键方面：(1)机械应变通过运动头传递以调节核苷酸亲和力的结构途径，这对运动头协调很重要；(2)微管c端柔性E-hook结构域在运动头轨迹偏移中的动态作用，这对运动蛋白如何迈出一步至关重要。这些问题将通过进一步的模拟得到彻底的研究。将产生突变的运动蛋白，专门改变模拟中发现的物理机制，并使用最先进的光学陷阱系统进行实验测试。这项工作的结果将提供一个更清晰的原子图像的运动力学基础的运动。以我们之前的r21资助项目为先导，将通过实验和模拟之间的强大协同作用来发展拟议的工作，这将成为我们长期目标研究其他许多运动蛋白的基础。公共卫生相关性：对运动蛋白运动的深入了解将有助于更好地控制其行为和运动特性，这将导致针对运动蛋白介导的运输的新疗法。我们将大分子复合物的计算建模和单分子操作实验相结合的方法提供了一个平台，在这个平台上，一系列具有生物医学重要性的亚细胞运动过程将被研究。

DESCRIPTION (provided by applicant): Kinesin is the smallest known biped motor protein that uses ATP as a fuel to walk processively along the microtubule track. Its proper function is critical for many vital tasks including intracellular cargo transport and cell division. A deeper insight into how kinesin functions is thus not only important for advancing fundamental knowledge of molecular motors, but also critical for developing novel therapeutics against diseases involving impaired intracellular transport. While past advances revealed many important aspects on its global motility characteristics, physical mechanism underling its stepping motion remains unclear. A major difficulty in studying kinesin motility or motor proteins in general, is that the molecule dynamically senses and generates force to move, which is difficult to contemplate based on static structural picture only. To investigate the dynamic aspect in atomistic detail, we take a synergistic approach between molecular dynamics simulation and single-molecule experiment. Using molecular dynamics simulations, we discovered that kinesin generates force by folding of a domain, which we named the cover-neck bundle. While the proposed mechanism is supported by our single-molecule motility experiments testing kinesin mutants designed to generate less force, the experiments led to further questions regarding energetics of the force generation as well as the role of the force-generating step in the overall kinesin mechanochemical cycle. Furthermore, our preliminary simulations identified two other crucial aspects of kinesin motility: (1) the structural pathway by which mechanical strain is transmitted through the motor head to modulate the nucleotide affinity, which is important for motor head coordination, and (2) the dynamic role of the C-terminal flexible E-hook domains of the microtubule in biasing the trajectory of a motor head, which is critical for how kinesin makes a step. These issues will be thoroughly investigated by further simulations. Mutant kinesins will be generated that specifically alter the physical mechanism found in simulations, and experimentally tested using state-of-the-art optical trap systems. Outcome of this work will provide a clearer atomistic picture of the mechanics underling kinesin motility. With our previous R21-funded project as a precursor, the proposed work will be developed via strong synergy between experiments and simulations, which will be the basis upon which a host of other motor proteins will be investigated as our long-term goal. PUBLIC HEALTH RELEVANCE: Deeper understanding of kinesin motility will enable better control of its behavior and motility characteristics, which will lead to novel therapeutics that target kinesin-mediated transport. Our combined approach of computational modeling of macromolecular complexes and single-molecule manipulation experiment provides a platform upon which a range of subcellular motor processes of biomedical importance will be investigated.