Kinesin's cover-​neck bundle folds forward to generate force

Each step of the kinesin motor involves a force-​generating mol. rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochem. cycle, mol. details of the force generation mechanism remain a mystery. Recent mol. dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-​terminal cover strand forms a β-​sheet with the neck linker to yield the cover-​neck bundle. We tested this proposal by comparing optical trapping motility measurements of cover strand mutants with the wild-​type. Motility data, as well as kinetic analyses, revealed impairment of the force-​generating capacity accompanied by a greater load dependence in the mechanochem. cycle. In particular, a mutant with the cover strand deleted functioned only marginally, despite the fact that the cover strand, the N-​terminal "dangling end,​" unlike the neck linker and nucleotide-​binding pocket, is not involved with any previously considered energy transduction pathway. Furthermore, a const. assisting load, likely in lieu of a power stroke, was shown to rescue forward motility in the cover strand deletion mutant. Our results support a stepping mechanism driven by dynamic cover-​neck bundle formation. They also suggest a strategy to generate motors with altered mech. characteristics by targeting the force-​generating element.