Structural transitions in kinesin minus-end directed microtubule motility
Kinesin motor proteins hydrolyze ATP to produce force for spindle assembly and vesicle transport, performing essential functions in cell division and motility, but the structural changes required for force generation are uncertain. We now report high-resolution structures showing new transitions in the kinesin mechanochemical cycle, including power stroke fluctuations upon ATP binding and a post-hydrolysis state with bound ADP + free phosphate. We find that rate-limiting ADP release occurs upon microtubule binding, accompanied by central {beta}-sheet twisting, which triggers the power stroke - stalk rotation and neck mimic docking - upon ATP binding. Microtubule release occurs with {beta}-strand-to-loop transitions, implying that {beta}-strand refolding induces Pi release and the recovery stroke. The strained {beta}-sheet during the power stroke and strand-to-loop transitions identify the {beta}-sheet as the long-sought motor spring. TeaserStalk rotation, {beta}-sheet twisting and refolding, and neck mimic docking drive the reversed working stroke of kinesin-14 INTRODUCTIONKinesin family proteins couple ATP hydrolysis to microtubule binding, generating force to produce steps or displacements along microtubules. The mechanism by which kinesins and other cytoskeletal motor proteins produce force is not fully understood. A current hypothesis is that the motors contain a spring-like or elastic element that creates strain under load during nucleotide binding or release, followed by a strain-relieving conformational change that produces force and a working stroke of the motor. The spring has not yet been identified for any motor. The power stroke differs for different motors - it consists of neck linker docking for plus-end directed kinesin-1 or a swing of the helical stalk for minus-end directed kinesin-14. RATIONALEDespite considerable research, the molecular dynamics of the kinesin-14 power stroke are still obscure, impeded by the weak microtubule binding of the motor. We overcame the weak binding by introducing a point mutation into the motor that results in faster ATP hydrolysis than wild type and tighter microtubule binding, which enabled us to resolve the motor mode of action. We now present high-resolution cryo-electron microscopy (cryo-EM) and x-ray structures of key mechanochemical states across the full force-producing cycle of a kinesin dimeric motor. RESULTSThe new structures represent five different nucleotide states - two pre-power stroke states, a fluctuating power stroke, and two post-power stroke states. The structures are both microtubule-attached and unattached. They show the motor trapped in previously unreported transition states and reveal new conformational changes involved in energy transduction. The new transition states include a transient state in which the power stroke fluctuates during ATP binding and a new state of a kinesin motor bound to ADP and free Pi prior to phosphate release. The conformational changes include the folding of the kinesin-14 neck mimic into a structure resembling the docked kinesin-1 neck linker, accompanying the power stroke, and previously unreported {beta}-strand-to-loop transitions with stored free energy that potentially induce Pi release and drive the recovery stroke. We interpret the new structures in the context of the hypothesis that the central {beta}-sheet undergoes distortional changes during the mechanochemical cycle that store and release free energy, functioning as the elusive spring of the motors. CONCLUSIONThe new structures show that force is produced by coupled movements of the helical stalk, central {beta}-sheet, and neck mimic, and uncover structural changes during the power stroke that are conserved among kinesins and myosin. We find that kinesin-14 binds to a microtubule by one head during the mechanical cycle, undergoes rate-limiting ADP release, and changes in conformation during ATP binding and hydrolysis to produce force. Notably, kinesin-14 utilizes the same mechanical strategy for force production as other kinesins but couples the changes to a large swing of the stalk, an innovation derived from myosin that is not observed for kinesin-1 or other kinesin motors. Force is produced by rearranging the binding surfaces of the stalk, strand {beta}1, helices [a]4 and [a]6, and the neck mimic, and by twisting and shortening strands of the central {beta}-sheet. These structural changes produce a power stroke - rotation of the helical stalk accompanied by neck mimic docking - during the transition from the nucleotide-free to ATP-bound state, and a reverse stroke after phosphate release that reprimes the motor for the next microtubule binding interaction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=97 SRC="FIGDIR/small/605428v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1093cdforg.highwire.dtl.DTLVardef@17b6c99org.highwire.dtl.DTLVardef@16b4a5org.highwire.dtl.DTLVardef@add967_HPS_FORMAT_FIGEXP M_FIG C_FIG Kinesin-14 force productionNew transition states and structural movements in a model for motor energy transduction and force production: {beta}-sheet twisting stores free energy in the microtubule-bound nucleotide-free (NF) state. A fluctuating power stroke is produced in the ATP state with neck mimic docking in the ADP{middle dot}Pi state, resembling the kinesin-1 neck linker. This is followed by {beta}-strand-to-loop transitions in the microtubule-bound ADP + free Pi state. Finally, {beta}-sheet refolding drives the recovery stroke for reversion to the ADP state.