Multivariate Data Analyses for Classifying Allosteric Inhibition in Human Eg5 Kinesin

Abstract

Like other motor proteins, the human Eg5 kinesin couples ATP hydrolysis to large conformational changes distal from the nucleotide site, thereby driving movement along microtubules. The Eg5 motor domain uniquely possesses an allosteric L5 loop, responsible for sensitivity to small-molecule inhibitors. Prior studies support a common kinetic mode of inhibition by monastrol, S-trityl-L-cysteine, and ispinesib. However, missing is the role of individual residues within the L5 loop in allosteric communication and why there are differing efficacies in drug inhibition. Here we demonstrate an integrated approach to build, test, and refine a model of how the L5 loop alters the conformation of the motor domain in solution. Over 30 perturbations of the L5 loop, either by sequence variation or drug binding, were analyzed using kinetic data, vibrational spectroscopy, and multivariate analysis. Principal component analysis organized mutant kinesins into two populations of Eg5 conformers, distinguished by changes mainly in 310 helices and unordered regions. The presence of inhibitors also resulted in coincident, steady-state structural changes in this kinesin. We surmise that the above conformational changes are localized to the L5 loop. Unexpectedly, conformational changes were not restricted to the drug-binding pocket alone: we have directly measured long-distance changes to the beta-sheet core of the kinesin protein, a requirement for allostery that is quantifiable in this analysis. Such tools can ultimately permit prediction of pleiotropic changes in structure and consequently protein function and drug efficacy.