Abstact

Kinesin motors use ATP to produce force in cells, yet the conformational changes that generate force remain uncertain. Here, we report structural and mechanistic insights into a minus-end-directed kinesin-14 that exhibits increased mechanical output - the variant motor binds microtubules more tightly and moves with faster velocity than wild type. High-resolution structures, together with molecular dynamics simulations, reveal previously unobserved transitions in the nucleotide hydrolysis cycle. ADP release, triggered by microtubule binding, is coupled to twisting of the central beta-sheet and stabilization of the stalk prior to the power stroke. ATP binding induces stalk fluctuations and a swing of the neck mimic, an element analogous to the kinesin-1 neck linker, resembling neck linker docking in plus-end-directed kinesins. The power stroke, characterized by a large stalk rotation, is followed by motor detachment from microtubules. The subsequent recovery stroke occurs while the motor is bound to ADP and free Pi, accompanied by beta-strand-to-loop transitions, or beta-sheet melting, implying that beta-sheet refolding facilitates Pi release. The observed twisting and melting identify the central beta-sheet as the long-sought elastic element or spring required for motor force production. The transitions we observe in kinesin-14 may also apply to other kinesins - this remains to be tested. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1038/s41598-025-28573-7.