Structure and function of Myosin VI

说明(申请人提供)：肌球蛋白VI可能是非常规肌球蛋白中最非常规的。它使用了许多独特的机制，这些机制还没有被很好地理解来完成与肌球蛋白V相似的步长的前进运动，但为了在细胞内锚定和运输的目的而具有相反的方向性。肌球蛋白VI可以以单体或二聚体的形式存在，其细胞意义尚不清楚。该项目将利用体外表达和功能分析、结构测定和体内分析，试图加深我们对肌球蛋白VI的功能以及这些分子在细胞中的作用的理解。这些目标将通过解决以下具体目标来实现：目标1.Myosin VI转换器中的哪些适应性可产生较大且可变(但固有的加尾方向)步长？假设：虽然肌球蛋白VI转化器末端的独特插入物，即插入物2，是肌球蛋白VI方向逆转的唯一原因，但肌球蛋白VI转化器具有额外的适应性，允许在PowerStroke前处于独特的状态，并产生较大且可变的步长。目的2.肌球蛋白VI杠杆臂+伸展异常的性质和目的是什么？假设：肌球蛋白VI的大步长部分是由于其转换器的不寻常运动和构象，这产生了比肌球蛋白V更大的摆动(PowerStroke)。在这种更大的摆动中，较短的杠杆臂可以提供与肌球蛋白V杠杆臂相同的步长。目的3.进一步探讨加工过程中头部门控的机制假说：肌球蛋白VI的唯一插入片段1负责缓慢的ATP结合，而反向应变使其变得更慢，而ADP可以接触到核苷酸结合口袋。这允许选通二聚体的引线头部，以及锚定二聚体。目的4.肌球蛋白VI是如何二聚化的，它在细胞中是作为二聚体还是作为单体发挥作用？(与Karen Avraham合作)假设：肌球蛋白VI的二聚化受货物相互作用的调节。我们认为，调节的二聚化消除了调节运动活动的需要。

DESCRIPTION (provided by applicant): Myosin VI is perhaps the most unconventional of unconventional myosins. It uses a number of unique mechanisms that are not well understood to accomplish processive movements of similar step sizes to myosin V, but of opposite directionality, for the purposes of anchoring and transporting within cells. Myosin VI can exist as a monomer or a dimer, the cellular significance of which is poorly understood. This project will utilize in vitro expression and functional assays, structural determinations and in vivo assays to attempt to further our understanding of how myosin VI functions and what these molecules do in a cell. These goals will be realized by addressing the following specific aims: Aim 1. What are the adaptations in the myosin VI converter that create a large and variable (but inherently plus-end directed) step size? Hypothesis: While the unique insert at the end of the myosin VI converter, insert 2, is solely responsible for the reversal of directionality of myosin VI, the myosin VI converter has additional adaptations that allow a unique pre-powerstroke state and create a large and variable step size. Aim 2. What is the nature and purpose of the unusual myosin VI lever arm + extension? Hypothesis: The large step size of myosin VI is in part due to the unusual movements and conformations of its converter, which creates a larger swing (powerstroke) than for myosin V. With this larger swing, a shorter lever arm can provide the same step size as the myosin V lever arm. Aim 3. Further probe the mechanism of head gating during processivity Hypothesis: The unique insert, insert 1, of myosin VI is responsible for slow ATP binding, which becomes still slower with reverse strain, while ADP has access to the nucleotide binding pocket. This allows gating of the lead head of a dimer, as well as anchoring of a dimer. Aim 4. How does myosin VI dimerize and does it function as a dimer or monomer in cells? (collaboration with Karen Avraham) Hypothesis: Myosin VI dimerization is regulated by cargo interactions. We propose that the regulated dimerization obviates the need to regulate motor activity.