title: Kinetics of nucleotide-dependent structural transitions in the kinesin-1 hydrolysis cycle.
abstract: 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 one-head-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.
authors.affiliation: Department of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802; Intercollege Graduate Degree Program in Bioengineering, Pennsylvania State University, University Park, PA 16802;
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Mickolajczyk
authors.affiliation: Department of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802; Intercollege Graduate Degree Program in Bioengineering, Pennsylvania State University, University Park, PA 16802;
authors.forename: Nathan C
authors.initials: NC
authors.lastname: Deffenbaugh
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, United Kingdom.
authors.forename: Jaime Ortega
authors.initials: JO
authors.lastname: Arroyo
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, United Kingdom.
authors.forename: Joanna
authors.initials: J
authors.lastname: Andrecka
authors.affiliation: Physical and Theoretical Chemistry Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3QZ, United Kingdom.
authors.forename: Philipp
authors.initials: P
authors.lastname: Kukura
authors.affiliation: Department of Biomedical Engineering, Pennsylvania State University, University Park, PA 16802; Intercollege Graduate Degree Program in Bioengineering, Pennsylvania State University, University Park, PA 16802; wohbio@engr.psu.edu.
authors.forename: William O
authors.initials: WO
authors.lastname: Hancock
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000378:metabolism; D000465:Algorithms; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008857:Microscopy, Interference; D008956:Models, Chemical; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D029721:Drosophila Proteins; D020409:Molecular Motor Proteins; D000255:Adenosine Triphosphate; D016547:Kinesins
keywords: iSCAT; kinesin; microscopy; structural kinetics; structure–function