Mechanisms of Processivity in Molecular Motors

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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.