Microtubule Binding And Rotation Of The Kinesin-14 Stalk

Movement of motors along cytoskeletal filaments is thought to be driven by small structural changes that are amplified by a large rotation of the alpha-helical coiled coil. The coiled-coil stalk of the kinesin-14 motor, Ncd, has been visualized in a rotated conformation in a crystal structure and proposed to act like a lever to amplify force produced by the motor, resulting in a working stroke that directs the motor to the microtubule minus end. We show here that an Ncd mutant that is trapped in a stalk-rotated conformation binds tightly to microtubules and shows fluorescence resonance energy transfer between the end of the stalk and microtubule, indicating that rotation of the stalk towards the microtubule is coupled to motor binding to microtubules. A mutant blocked in stalk rotation binds weakly to microtubules and shows no energy transfer, demonstrating that binding by the motor to microtubules requires movement of the stalk. Energy transfer assays show that wild-type Ncd binds to microtubules without added nucleotide with the end of the stalk more than ∼9 nm from the microtubule, rotating less than 50 degrees from a position perpendicular to the microtubule. Upon binding with a non-hydrolysable ATP analogue, the stalk lies within ∼6 nm of the microtubule surface, representing a rotation of ∼70 degrees. These findings are consistent with previous reports by cryoEM that the Ncd stalk rotates when the microtubule-bound motor binds ATP. However, our results indicate that stalk rotation is initiated by filament binding and ADP release, and completed upon ATP binding, rather than triggered by ATP binding. Initiation of the Ncd stalk rotation by microtubule binding and ADP release, and its completion on ATP binding is reminiscent of the two-step working stroke of myosin I, revealing an unexpected similarity between the motors.