Understanding the Role of the Conformational Changes in the Kinesin-5 on Processivity and Inhibition
Startup Project (May 1, 2018 - April 30, 2020)
Human Kinesin-5, an anticancer drug target, is key to the assembly of the bipolar spindle during mitosis. This research project seeks to define the mechanism of mechanical output by kinesin motor proteins. Our interest is in the neck-linker, a 12-15 residue segment at the N-terminus of kinesin plays an important role in processivity. However, it is not well understood the importance of the conformational change that forms a short β-sheet from a floppy region to its inhibition by anticancer drugs nor its influence on the coil-coil domain, the region necessary for oligomerization. The goal of this project is to determine the effects of substitutions and insertions in the neck-linker of kinesin-5, including human polymorphic variants, on in vitro measurements of catalytic activity and motion and whether the neck-linker controls structural asymmetry and initiation of the coil-coil, necessary for kinesin oligomerization. AIM1: We will insert three residues (DAL) into various places along the neck-linker of dimeric Kinesin-5 construct (Eg5-513) followed by expression and purification of the mutants in a bacterial system. We predict that the loss of the neck-linker will start the result in lower processivity of the chimera as the conformational change. Our experimental readout will be MT gliding by the mutant Kinesin-5. We will quantitate MT-gliding velocities for each mutant Kinesin-5 motor as well as WT Kinesin-5 for control. We expect that perturbations to the individual β-strands will prevent the extension of the central β-sheet by the cover neck bundle and reduce the motility of the motor along MT tracks. The anticipated result is that we expect a loss of interaction between β7 and β10 as a result of the glycine mutations. We anticipate that this will lead to total loss of kinesin motility. We will then test the response to inhibition by current anticancer inhibitor STLC AIM 2: We will generate mutations to the dimeric Kinesin-5 construct (Eg5-513) in β7 (E254G, L255G, V256G, K257G, I258G) by site-directed mutagenesis followed by expression and purification of the mutants in a bacterial system. It has been ignored that the docking of the NL and cover-neck to the motor head coordinates an interaction with β7. We will test the inhibitory effects of these mutations with the inhibitor STC on ATPase activity and processivity. The result of these experiments will provide a framework for understanding the role of the neck-linker as well as the cover neck bundle in Eg5 and may be expended to other kinesins and motor proteins which also exhibit conformational switching.