title: Processivity of the kinesin-2 KIF3A results from rear head gating and not front head gating.
abstract: The kinesin-2 family motor KIF3A/B works together with dynein to bidirectionally transport intraflagellar particles, melanosomes, and neuronal vesicles. Compared with kinesin-1, kinesin-2 is less processive, and its processivity is more sensitive to load, suggesting that processivity may be controlled by different gating mechanisms. We used stopped-flow and steady-state kinetics experiments, along with single-molecule and multimotor assays to characterize the entire kinetic cycle of a KIF3A homodimer that exhibits motility similar to that of full-length KIF3A/B. Upon first encounter with a microtubule, the motor rapidly exchanges both mADP and mATP. When adenosine 5'-[(β,γ)-imido]triphosphate was used to entrap the motor in a two-head-bound state, exchange kinetics were unchanged, indicating that rearward strain in the two-head-bound state does not alter nucleotide binding to the front head. A similar lack of front head gating was found when intramolecular strain was enhanced by shortening the neck linker domain from 17 to 14 residues. In single-molecule assays in ADP, the motor dissociates at 2.1 s(-1), 20-fold slower than the stepping rate, demonstrating the presence of rear head gating. In microtubule pelleting assays, the KD(Mt) is similar in ADP and ATP. The data and accompanying simulations suggest that, rather than KIF3A processivity resulting from strain-dependent regulation of nucleotide binding (front head gating), the motor spends a significant fraction of its hydrolysis cycle in a low affinity state but dissociates only slowly from this state. This work provides a mechanism to explain differences in the load-dependent properties of kinesin-1 and kinesin-2.
authors: From the Department of Biomedical Engineering Pennsylvania State University University Park, Pennsylvania 16802.
authors: Geng-Yuan
authors: GY
authors: Chen
authors: From the Department of Biomedical Engineering Pennsylvania State University University Park, Pennsylvania 16802.
authors: David F J
authors: DF
authors: Arginteanu
authors: From the Department of Biomedical Engineering Pennsylvania State University University Park, Pennsylvania 16802 wohbio@engr.psu.edu.
authors: William O
authors: WO
authors: Hancock
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006868:Hydrolysis; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008870:Microtubules; D008969:Molecular Sequence Data; D009038:Motion; D020033:Protein Isoforms / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029721:Drosophila Proteins; C490202:Kif3a protein, mouse; C490672:Kif3b protein, mouse; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D000255:Adenosine Triphosphate; C515209:Khc protein, Drosophila; D016547:Kinesins
keywords: Kinesin, Microtubule, Intracellular Trafficking, Enzyme Kinetics, Single-molecule Biophysics, Molecular Motor, Cilia
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