Kinesin's cover-neck bundle folds forward to generate force.
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.
Khalil
Ahmad S
AS
Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Appleyard
David C
DC
Labno
Anna K
AK
Georges
Adrien
A
Karplus
Martin
M
Belcher
Angela M
AM
Hwang
Wonmuk
W
Lang
Matthew J
MJ
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
D029721:Drosophila Proteins; D016547:Kinesins