Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains.
Kinesin is a processive motor protein: A single molecule can walk continuously along a microtubule for several micrometers, taking hundreds of 8-nm steps without dissociating. To elucidate the biochemical and structural basis for processivity, we have engineered a heterodimeric one-headed kinesin and compared its biochemical properties to those of the wild-type two-headed molecule. Our construct retains the functionally important neck and tail domains and supports motility in high-density microtubule gliding assays, though it fails to move at the single-molecule level. We find that the ATPase rate of one-headed kinesin is 3-6 s(-1) and that detachment from the microtubule occurs at a similar rate (3 s(-1)). This establishes that one-headed kinesin usually detaches once per ATP hydrolysis cycle. Furthermore, we identify the rate-limiting step in the one-headed hydrolysis cycle as detachment from the microtubule in the ADP.P(i) state. Because the ATPase and detachment rates are roughly an order of magnitude lower than the corresponding rates for two-headed kinesin, the detachment of one head in the homodimer (in the ADP.P(i) state) must be accelerated by the other head. We hypothesize that this results from internal strain generated when the second head binds. This idea accords with a hand-over-hand model for processivity in which the release of the trailing head is contingent on the binding of the forward head. These new results, together with previously published ones, allow us to propose a pathway that defines the chemical and mechanical cycle for two-headed kinesin.
Hancock
W O
WO
Department of Physiology, University of Washington, Seattle, WA, 98195-7290, USA.
Howard
J
J
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D004330:Drosophila; D006868:Hydrolysis; D016547:Kinesins / Q000378:metabolism*; D007700:Kinetics; D008870:Microtubules / Q000378:metabolism
D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins