Lever Arm Mobility and Force Generation in Ncd, a Minus-End Kinesin Motor

Abstract

Unlike the conventional kinesin motor proteins, which step towards the plus end of the microtubule, the kinesin-14 family translocates cargo towards microtubule minus ends. Following microtubule attachment, kinesin-14 motors use energy derived from ATP hydrolysis to swing a arm, consisting of a long coiled-coil dimerization domain, in order to move their cargo in the direction of travel. While atomic-resolution structures of several kinesin-14 constructs have been solved, the active states of force transduction only occur when the motor is attached to the microtubule. Thus, the mechanism that drives lever arm movement remains unclear. We solved a series of sub-nanometer cryo-EM reconstructions of dimeric ncd, a kinesin-14 family member, in different chemical states while attached to the microtubule. The maps reveal a series of well-defined structural rearrangements in the ncd motor domain, suggesting a mechanism for how microtubule attachment and subsequent ATP binding can trigger a lever arm swing. Unexpectedly, our maps also indicate that the unbound (or “tethered”) catalytic head of our dimeric ncd construct, which does not contact the microtubule, remains rotationally mobile in all the microtubule-attached nucleotide states we examined. In contrast, the other head always attaches to the microtubule in a well-defined orientation, and the lever arm of the motor assembly projects rigidly away from this head while exhibiting either “pre-” or “post-” power stroke conformations. These results suggest a model in which the catalytic domains of an unattached ncd dimer oscillate between different lever arm states in order to facilitate the search for a new microtubule binding site; attachment would then lock the lever arm angle in preparation for a power stroke.