title: Kinesin's cover-neck bundle folds forward to generate force.
abstract: Each step of the kinesin motor involves a force-generating molecular rearrangement. Although significant progress has been made in elucidating the broad features of the kinesin mechanochemical cycle, molecular details of the force generation mechanism remain a mystery. Recent molecular dynamics simulations have suggested a mechanism in which the forward drive is produced when the N-terminal cover strand forms a beta-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 mechanochemical 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 constant 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 mechanical characteristics by targeting the force-generating element.
authors.affiliation: Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Ahmad S
authors.initials: AS
authors.lastname: Khalil
authors.forename: David C
authors.initials: DC
authors.lastname: Appleyard
authors.forename: Anna K
authors.initials: AK
authors.lastname: Labno
authors.forename: Adrien
authors.initials: A
authors.lastname: Georges
authors.forename: Martin
authors.initials: M
authors.lastname: Karplus
authors.forename: Angela M
authors.initials: AM
authors.lastname: Belcher
authors.forename: Wonmuk
authors.initials: W
authors.lastname: Hwang
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Lang
mesh_terms: D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D016296:Mutagenesis; D052898:Optical Tweezers; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D029721:Drosophila Proteins; D016547:Kinesins