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.
Molecular motors drive diverse forms of cell physiology. In this application, I propose to investigate how two members of the kinesin family of molecular motors adjust their structures in order to adapt their enzymologies to the specific demands placed on them by the cell. I anticipate that this work will illuminate design principles for understanding how existing motors work as well as generate insights into how new motors could be custom engineered to serve specific functions.
|Atherton, Joseph; Farabella, Irene; Yu, I-Mei et al. (2014) Conserved mechanisms of microtubule-stimulated ADP release, ATP binding, and force generation in transport kinesins. Elife 3:e03680|
|Goulet, Adeline; Major, Jennifer; Jun, Yonggun et al. (2014) Comprehensive structural model of the mechanochemical cycle of a mitotic motor highlights molecular adaptations in the kinesin family. Proc Natl Acad Sci U S A 111:1837-42|
|Kaan, Hung Yi Kristal; Major, Jennifer; Tkocz, Katarzyna et al. (2013) "Snapshots" of ispinesib-induced conformational changes in the mitotic kinesin Eg5. J Biol Chem 288:18588-98|