TY - JOUR
T1 - Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle
AU - Mickolajczyk, Keith J.
AU - Deffenbaugh, Nathan C.
AU - Arroyo, Jaime Ortega
AU - Andrecka, Joanna
AU - Kukura, Philipp
AU - Hancock, William O.
N1 - Funding Information:
We thank David Arginteanu for his help with protein preparation and purification, the laboratory of Randy Schekman for providing plasmids, and John Fricks and members of W.O.H.''s laboratory for helpful discussions. This work was funded by NIH Grant R01 GM076476 (to W.O.H.) and a European Research Council starting grant (NanoScope) (to P.K.). J.O.A. was supported by a scholarship from Consejo Nacional de Ciencia y Tecnología (Scholar 213546), and J.A. was supported by Marie Curie Fellowship 330215.
PY - 2015/12/29
Y1 - 2015/12/29
N2 - To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the onehead-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.
AB - To dissect the kinetics of structural transitions underlying the stepping cycle of kinesin-1 at physiological ATP, we used interferometric scattering microscopy to track the position of gold nanoparticles attached to individual motor domains in processively stepping dimers. Labeled heads resided stably at positions 16.4 nm apart, corresponding to a microtubule-bound state, and at a previously unseen intermediate position, corresponding to a tethered state. The chemical transitions underlying these structural transitions were identified by varying nucleotide conditions and carrying out parallel stopped-flow kinetics assays. At saturating ATP, kinesin-1 spends half of each stepping cycle with one head bound, specifying a structural state for each of two rate-limiting transitions. Analysis of stepping kinetics in varying nucleotides shows that ATP binding is required to properly enter the onehead-bound state, and hydrolysis is necessary to exit it at a physiological rate. These transitions differ from the standard model in which ATP binding drives full docking of the flexible neck linker domain of the motor. Thus, this work defines a consensus sequence of mechanochemical transitions that can be used to understand functional diversity across the kinesin superfamily.
UR - http://www.scopus.com/inward/record.url?scp=84952673485&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84952673485&partnerID=8YFLogxK
U2 - 10.1073/pnas.1517638112
DO - 10.1073/pnas.1517638112
M3 - Article
C2 - 26676576
AN - SCOPUS:84952673485
SN - 0027-8424
VL - 112
SP - E7186-E7193
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 52
ER -