The Molecular Mechanism of the Multi-Tasking Kinesin-8 Motor

Abstract

The mitotic spindle is organised by many microtubule-binding proteins including kinesin molecular motors. Depletion of kinesin-8 motors causes mitotic defects and long spindle microtubules, suggesting that kinesin-8s play a role in microtubule length regulation. Kinesin-8 motors have the remarkable ability to both walk towards microtubule plus-ends and to depolymerise these ends on arrival, which results in length-dependent depolymerisation.To understand how kinesin-8s achieve such multi-tasking, we studied the structure and function of the human kinesin-8 motor domain. We determined its crystal structure which is similar to that of the well-characterised depolymerising motors, the kinesin-13s. Both families of depolymerisers have an extended loop2, which is disordered in the kinesin-8 structure, and consistent with this, our kinesin-8 construct had depolymerising activity. However, its ATPase was stimulated by both tubulin - typical of a depolymeriser - and microtubule polymers - expected for a motile kinesin - demonstrating that the ability of kinesin-8s to both walk along microtubules and to depolymerise them is intrinsic to their motor domain. To help understand this functional paradox, we used cryo-electron microscopy to image microtubules bound by the kinesin-8 motor domain in nucleotide-free and ATP-like states and calculated the structures of these complexes at ∼10A resolution. Docking of the kinesin-8 crystal structure in the reconstructions revealed microtubule-dependent conformations of the motor. Strikingly, our reconstructions show that kinesin-8 loop2 provides an additional point of contact to the microtubule surface and likely contributes to the kinesin-8 processivity that is an essential aspect of their length regulatory activity. From our functional and structural studies we conclude that the kinesin-8s represents a unique family of microtubule depolymerizers and we can begin to dissect the molecular mechanism by which they act to control microtubule length and dynamics.