Computational and single molecule analysis of kinesin's atomistic machinery

驱动蛋白原子机制的计算和单分子分析

• 批准号: 7920016
• 负责人: Wonmuk Hwang
• 金额: $ 22.56万
• 依托单位: TEXAS ENGINEERING EXPERIMENT STATION
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-09-01 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7920016
• 关键词: ATP phosphohydrolase; Address; Adenosine Triphosphate; Affect; Affinity; Arts; Be++ element; Behavior; Beryllium; Binding; Binding Sites; C-terminal; Cell division; Characteristics; Computer Simulation; Diffusion; Disease; Docking; Elements; Event; Free Energy; Funding; Generations; Goals; Head; Intracellular Transport; Kinesin; Kinetics; Knowledge; Lead; Macromolecular Complexes; Measures; Mechanics; Mediating; Microtubules; Modeling; Molecular Motors; Motion; Motor; Mutation; N-terminal; Names; Neck; Nucleotides; Outcome; Pathway interactions; Power stroke; Process; Proteins; Regulation; Role; Series; Specificity; Spectrum Analysis; System; Testing; Thermodynamics; Tubulin; Walking; Work; base; cell motility; design; dimer; flexibility; insight; molecular dynamics; mutant; new therapeutic target; novel therapeutics; optical traps; post stroke; public health relevance; research study; simulation; single molecule; success

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.

描述（由申请人提供）：驱动蛋白是已知最小的双足运动蛋白，它使用 ATP 作为燃料沿着微管轨道持续行走。它的正确功能对于许多重要任务至关重要，包括细胞内货物运输和细胞分裂。因此，更深入地了解驱动蛋白的功能不仅对于推进分子马达的基础知识很重要，而且对于开发针对细胞内运输受损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，研究驱动蛋白运动或运动蛋白的一个主要困难是分子动态感知并产生运动力，这很难仅基于静态结构图来考虑。为了研究原子细节的动态方面，我们采用分子动力学模拟和单分子实验之间的协同方法。通过分子动力学模拟，我们发现驱动蛋白通过折叠域（我们将其命名为盖颈束）来产生力。虽然所提出的机制得到了我们的单分子运动实验的支持，该实验测试了旨在产生较小力的驱动蛋白突变体，但这些实验引发了有关力产生的能量学以及力产生步骤在整个驱动蛋白机械化学中的作用的进一步问题循环。此外，我们的初步模拟还确定了驱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，这对于运动头协调很重要；（2）动态微管 C 端柔性 E-hook 结构域在偏置运动头轨迹中的作用，这对于驱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进行彻底研究。将产生突变的驱动蛋白，专门改变模拟中发现的物理机制，并使用最先进的光陷阱系统进行实验测试。这项工作的成果将为驱动蛋白运动机制提供更清晰的原子图。以我们之前的 R21 资助项目为先导，拟议的工作将通过实验和模拟之间的强大协同作用进行开发，这将成为我们长期目标研究许多其他运动蛋白的基础。公共健康相关性：对驱动蛋白运动的更深入了解将能够更好地控制其行为和运动特征，这将导致针对驱动蛋白介导的运输的新疗法。我们的大分子复合物计算模型和单分子操作实验的组合方法提供了一个平台，在此平台上将研究一系列具有生物医学重要性的亚细胞运动过程。