title: The Kinesin-5 Chemomechanical Cycle Is Dominated by a Two-heads-bound State.
abstract: Single-molecule microscopy and stopped-flow kinetics assays were carried out to understand the microtubule polymerase activity of kinesin-5 (Eg5). Four lines of evidence argue that the motor primarily resides in a two-heads-bound (2HB) state. First, upon microtubule binding, dimeric Eg5 releases both bound ADPs. Second, microtubule dissociation in saturating ADP is 20-fold slower for the dimer than for the monomer. Third, ATP-triggered mant-ADP release is 5-fold faster than the stepping rate. Fourth, ATP binding is relatively fast when the motor is locked in a 2HB state. Shortening the neck-linker does not facilitate rear-head detachment, suggesting a minimal role for rear-head-gating. This 2HB state may enable Eg5 to stabilize incoming tubulin at the growing microtubule plus-end. The finding that slowly hydrolyzable ATP analogs trigger slower nucleotide release than ATP suggests that ATP hydrolysis in the bound head precedes stepping by the tethered head, leading to a mechanochemical cycle in which processivity is determined by the race between unbinding of the bound head and attachment of the tethered head.
authors.affiliation: From the Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Geng-Yuan
authors.identifier: 0000-0002-0103-4494
authors.initials: GY
authors.lastname: Chen
authors.affiliation: From the Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802.
authors.forename: Keith J
authors.initials: KJ
authors.lastname: Mickolajczyk
authors.affiliation: From the Department of Biomedical Engineering, Pennsylvania State University, University Park, Pennsylvania 16802 wohbio@engr.psu.edu.
authors.forename: William O
authors.identifier: 0000-0001-5547-8755
authors.initials: WO
authors.lastname: Hancock
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins; D000072417:Protein Domains; D029867:Xenopus Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D029721:Drosophila Proteins; C088335:KIF11 protein, Xenopus; C084034:Klp61F protein, Drosophila; D008869:Microtubule-Associated Proteins; D029867:Xenopus Proteins; D000255:Adenosine Triphosphate; D016547:Kinesins
keywords: cytoskeleton; kinesin; microtubule; mitotic spindle; molecular motor; single-molecule biophysics