Kinesin-8 motors dimerize by folding their proximal tail domain into a compact helical bundle

Kinesin-8 motor proteins help align and segregate chromosomes during mitosis by regulating the dynamics of kinetochore-attached microtubules and the length and position of the mitotic spindle. Some kinesin-8 isoforms accomplish these roles by operating as multifunctional mechanoenzymes that can traverse microtubules, accumulate at the microtubule plus-ends, and then remove terminal αβ-tubulin subunits. While these activities are mainly powered by the motor domain, whose unique structure-function relationships have been recently reported, the non-motor tail domain contains integral functional elements that have not been structurally illuminated. Using the Candida albicans Kip3 protein as a kinesin-8 model system, we present an X-ray crystal structure and hydrodynamic data showing how the motor domain-proximal segment of the tail directs the assembly of two kinesin-8 polypeptides into a homodimer that forms the stalk of this motor. Unlike the extended coiled coil-forming helices of most other motile kinesin stalks, the proximal tail of CaKip3 folds into a compact 92 Å-long four-helix bundle that dimerizes. The first and third helices provide most of the surface area for the dimer interface, while the other two helices brace the folded stalk structure. The upper and lower lobules of the helical bundle are separated by a flexible hinge that gives the exterior faces of the stalk slightly different shapes when bent. We propose that these unique characteristics provide structural rigidity to the kinesin-8 stalk, as well as sites for transient interactions with kinesin-8-associated proteins or other regulatory regions of the motor.