Noncanonical Microtubule Interaction of Yeast Kinesin-5 CIN8

Abstract

Kinesin-5's were originally described as slow, plus-end-directed motors that are required for proper establishment and maintenance of the mitotic spindle. They share a bipolar homotetrameric structure with four heavy chains organized such that two motor domains are positioned at each end of a coiled-coil stalk, allowing the motor to cross-link and slide two antiparallel microtubules. Interestingly, recent studies have demonstrated bidirectional motility for yeast kinesin-5 motors, which represents a significant biophysical conundrum given our previous understanding of directed kinesin motility. We aim to understand the structural and biochemical features of yeast kinesin-5 motors that promote directional switching of motility. Saccharomyces cerevisiae Cin8 has been shown to switch directionality based on ionic strength, motor coupling and binding between antiparallel microtubules. Using ionic strength to promote switching of directionality, we have determined the microtubule binding behavior, steady-state kinetics, and cryo-EM structure of the Cin8 motor domain. Cosedimentation assays revealed that Cin8 binds super-stoichiometrically along the microtubule lattice with 3±1 motor domains binding per tubulin dimer. Competition assays with human kinesin-5 (Eg5) indicate that Cin8 binds at the canonical kinesin binding site as well as at a yet unidentified non-canonical site. Interestingly, Cin8 has microtubule-stimulated ATPase activity on both the canonical and non-canonical sites. We have probed the structural explanation for these biochemical observations by obtaining a 7A cryo-EM reconstruction of the microtubule-Cin8 complex in the ADP+AlFx state. Deletion of the large insert in Cin8's loop L8 retains similar steady-state ATPase activity yet abolishes super-stoichiometric microtubule binding, providing a powerful tool to dissect its ATPase mechanism. Cin8's microtubule interaction offers a mechanism for bidirectional movement, such that the motor utilizes a non-canonical microtubule site for directional switching.