Structural Basis for Nucleotide Exchange and Power Stroke Generation by the Kinesin Molecular Motor

Kinesin molecular motors use energy derived from ATP to step along microtubules, driving many essential processes in eukaryotic cells, including mitosis, vesicle transport and cytoskeletal remodeling. Several conformational states of kinesin have been identified by X-ray crystallography, but the structural transitions used by kinesin to generate force and movement along intact microtubules have remained unclear. We used recent improvements in cryo-EM methodology and instrumentation to capture the conformation of microtubule-attached kinesin at the beginning (no-nucleotide) and end (ATP analog-bound) of the force generation process at 5-6A resolution. We derived all-atom models for these two maps from a crystal structure of the tubulin-kinesin complex, using explicitly solvated molecular dynamics simulations combined with restraints derived from the maps. This analysis revealed that, contrary to existing models, kinesin's central beta sheet serves as the primary transducer in the motor's force-generation mechanism, twisting to drive ADP release and subsequently untwisting upon ATP binding to trigger a power stroke. We identified conserved residues on the motor domain, supported by additional structural and biochemical analysis of site-directed mutations, which serve as allosteric latches during the motor's microtubule-attached phase. These latches regulate the sheet-twisting motion and couple key properties of motor function to each other, including nucleotide binding, hydrolysis, and generation of a power stroke. These findings reveal how interactions with the microtubule can fundamentally alter kinesin's energetic landscape in order to initiate productive motility.