MYOSIN VI STEPS BACKWARDS: A COMPUTATIONAL STUDY TO ELUCIDATE THE MECHANISM

描述(申请人提供)：肌球蛋白是普遍存在的细胞内马达，它利用ATP水解的能量在肌动蛋白细丝上运动。自从肌球蛋白II首次在脊椎动物肌肉中被发现后，各种其他类别的肌球蛋白也被发现。最近有研究表明，VI类肌球蛋白在肌动蛋白上“向后”行走，即与其他肌球蛋白的方向相反。随后的研究揭示了这种反向运动在各种细胞中的重要运输和锚定作用。最近已经获得了几种不同构型的电机的晶体结构。在这个方案中，我们打算利用这些结构和其他实验数据，通过数值模拟的方法，在原子水平上阐明肌球蛋白VI马达的完整周期和能量。我们将使用最小能量路径(MEP)、块简正模式(BNM)分析和定向/偏向/靶向分子动力学来确定可用晶体结构之间的过渡路径。将计算与跃迁相关的平均力的势，这将给出与跃迁相关的自由能变化。我们将通过自由能模拟方法确定不同配体的晶体结构的亲和力，以确定不同的配体如何稳定不同的构象。为了了解肌球蛋白VI和其他(向前推进的)肌球蛋白之间的差异，我们将比较肌球蛋白VI和肌球蛋白V的机制(对肌球蛋白V的研究目前正在Karplus实验室进行)。上述许多方法都已在CHARMM程序中实现，我们将使用该程序进行模拟和分析。结合我们对运动头的分析，结合现有的肌球蛋白复合体的冷冻电子显微镜结构，文献中的单分子研究，以及弹性理论，我们将为这种不寻常的肌球蛋白开发一个粗粒度模型。 肌球蛋白VI功能障碍与遗传性听力损失有关，卵巢癌细胞中肌球蛋白VI浓度远高于正常细胞。我们的研究结果可能会导致针对这些疾病的新疗法的设计。例如，了解肌球蛋白VI的机制可以确定调节该马达活动的方法。

DESCRIPTION (provided by applicant): Myosins are ubiquitous intracellular motors that use the energy of ATP hydrolysis to move on actin filaments. After myosin II was first characterized in vertebrate muscle, various other classes have been found. Class VI myosin has been shown recently to walk "backwards" on actin, i.e. in the direction opposite to that of other myosins. Subsequent studies revealed important transport and anchoring roles for this backward motor in a variety of cells. Several crystal structures of the motor in various conformations have been obtained recently. In this proposal, we intend to utilize these structures and other experimental data to elucidate the complete cycle and energetics of the myosin VI motor at the atomic level by means of numerical simulations. We will determine transition paths between the available crystal structures using Minimum Energy Path (MEP), Block Normal Mode (BNM) analysis, and steered/biased/targeted Molecular Dynamics. The potential of mean force associated with the transitions will be computed, which will give the free energy change associated with the transitions. We will determine the affinities of the crystal structures for the various ligands by free energy simulation methods to determine how different conformations are stabilized by the different ligands. To understand the differences between myosin VI and other (forward-stepping) myosins, we will compare the mechanisms of myosin VI and myosin V (research on the myosin V is currently being done in the Karplus laboratory). Many of the methods mentioned above have been implemented in the program CHARMM, which we will use for simulation and analysis. Combining our analysis of the motor head with available cryo-electron microscopy structures of the acto-myosin complex, single-molecule studies in the literature, and elasticity theory, we will develop a coarse-grained model for this unusual myosin. Myosin VI malfunction is associated with hereditary hearing loss, and myosin VI concentration is much higher in ovarian cancer cells than in normal cells. Results of our research could lead to the design of new therapies for these diseases. For example, an understanding of the mechanism of myosin VI can identify ways to regulate the activity of this motor.