Mechanisms of Processivity in Molecular Motors

描述（由申请人提供）：在上一个资助期内，我发现肌凝蛋白V、肌凝蛋白VI、肌动蛋白1和CENP-E——进化上不同但功能上相似的分子马达——各自以相同的方式协调其两个催化结构域（“头部”）的酶学：通过控制核苷酸与前导头部的结合。在这个更新申请中，我现在希望进一步探讨这个变构通信的问题，这次是通过研究来自同一家族但具有不同功能的两个马达如何在一个头部内（分子内）以及头部之间（分子间）进行通信。Kinesin 1作为单个马达运输货物，在其微管（MT）轨道上行驶超过100步而不解离。Eg5在整体中滑动反平行主轴mt，对抗ncd和dynein的持续对立力；而且每次进程运行平均只需要8步。这些功能差异反映在不同的酶学上。与激酶1不同，ATP与Eg5的结合是缓慢的，并且与颈连接子对接紧密耦合。我将重点讲三个结构

DESCRIPTION (provided by applicant): In the last funding period, I found that myosin V, myosin VI, kinesin 1, and CENP-E--evolutionarily diverse but functionally similar molecular motors--each coordinate the enzymology of their two catalytic domains ("heads") in the same way: by gating nucleotide binding to the leading head. In this renewal application, I now wish to explore this issue of allosteric communication further, this time by investigating how two motors that come from the same family but serve different functions communicate--both within one head (intra- molecularly) as well as between heads (inter-molecularly). Kinesin 1 transports cargoes as a single motor, taking greater than 100 steps on its microtubule (MT) track without dissociating. Eg5 slides anti-parallel spindle MTs in ensembles, working against sustained opposing forces from ncd and dynein; and it only takes on average 8 steps per processive run. These functional differences are reflected in different enzymologies. Unlike kinesin 1, ATP binding to Eg5 is slow and tightly coupled to neck linker docking. I will focus on three structures that vary considerably between these two motors and which I propose play key roles in mediating both intra- and inter-molecular communication. These are loop L5, the neck linker, and the neck coiled coil. In Aim 1, I will examine how loop L5 regulates the timing of nucleotide binding and coupling to movements of the mechanical element--the neck linker. Experiments in this aim will utilize state- of-the-art transient kinetic and spectroscopic methodologies. In Aim 2 I will examine how polymorphisms in the neck linker and the neck-coiled coil contribute to differences in motor processivity. This work will combine the state-of-the-art methodologies developed in Aim 1 with single molecule mechanical studies. Taken together, Aims 1 and 2 should lead to the development of a comprehensive model of how kinesin motors "fine tune" their molecular physiology by adjusting a discrete number of structures. Kinesin 1 dysfunction has been linked to a number of neuro-degenerative diseases and to chemotherapy resistance in a variety of malignancies, and Eg5 has been intensively investigated as a target for the development of new anti-mitotics for the treatment of cancer. It is therefore likely that a molecular level model of how motors function will not only impact our understanding of pathophysiology but also point to new therapeutic approaches.