A Structural Model Of The Mitotic Kinesin-6 Mechanochemical Cycle

MKLP2 is a member of the kinesin-6 family, with critical roles during the metaphase-anaphase transition and in cytokinesis. The MKLP2 motor domain contains the conserved nucleotide and microtubule binding sites characteristic of plus-end mitotic kinesins. However, it is ∼60% larger than most other kinesins due to large inserts, the precise role(s) of which are unknown.Our biochemical studies show that the MKLP2 motor domain has nucleotide-insensitive sub-micromolar affinity for microtubules, except in the presence of the ATP analogue ADP.AlFx, which induces apparent nanomolar affinity. Consistently, we find that ATP binding kinetics appear to be uncoupled from microtubule binding. Using cryo-electron microscopy and structure determination (to ∼5A resolution), we have visualized the microtubule-bound MKLP2 motor domain at successive steps in its ATPase cycle. Its orientation with respect to the microtubule surface is conserved compared to other kinesins. ADP release induces small perturbations of the P-loop and loop11, described for transport kinesins and consistent with high levels of conservation in these regions. However, sequence divergence within the motor manifests itself at larger scales. Strikingly, ADP release does not induce major conformational changes around the nucleotide-binding site. However, in the presence of the ADP.AlFx, loop11 and loop9 are ordered, becoming compact around the bound nucleotide, forming a hydrolysis-competent conformation. The divergent loop2 also forms a contact with the microtubule surface, as do regions of the highly extended loop6. Along with these changes, the elongated kinesin-6 neck-linker is directed towards the microtubule plus-end but does not dock along the motor domain. The N-terminus of the motor is also disordered in all states and a cover neck bundle is not formed.Our data contribute to the mechanochemical dissection of this divergent mitotic kinesin and emphasize the critical mechanistic contribution of the motor interface with the microtubule.