Evidence for Motility Determinants in the Kinesin-1 Minimal Motor Core Domain From Tether Variations.

Kinesin-1 is a dimeric motor protein that moves towards the microtubule plus-end in a hand-over-hand fashion. However, the minimal motor domain of kinesin-1 is a single head, and the mechanism by which minimal motor domains generate the force for directional movement remains poorly understood. Here, we engineered artificial tethers (polyethylene glycol, single-stranded DNA, or double-stranded DNA) within the motor domain to investigate whether tether properties such as charge, length, and stiffness affect the motility of teams of kinesin-1 monomers. Neck-linker tethered kinesin with long stiff tethers was found to decrease microtubule-gliding velocity in an in vitro gliding assay, indicating that amplified conformational changes in the neck-linker do not enhance motility. Loop-12 tethered kinesin monomers with various tethers showed consistent minus-end-directed motility, reversing the usual polarity of kinesin-1 monomers. Moreover, loop-3 tethered kinesin monomers switched their directionality depending on tether stiffness. These results indicate that the tether has the potential to influence the direction in which the minimal motor domain moves. We argue that the determinants of motility exist in the minimal motor domain, with the combination of tether properties and its attachment position altering the MT-gliding velocity and direction.