Rapid double 8-nm steps by a kinesin mutant.
The mechanism by which conventional kinesin walks along microtubules is poorly understood, but may involve alternate binding to the microtubule and hydrolysis of ATP by the two heads. Here we report a single amino-acid change that affects stepping by the motor. Under low force or low ATP concentration, the motor moves by successive 8-nm steps in single-motor laser-trap assays, indicating that the mutation does not alter the basic mechanism of kinesin walking. Remarkably, under high force, the mutant motor takes successive 16-nm displacements that can be resolved into rapid double 8-nm steps with a short dwell between steps, followed by a longer dwell. The alternating short and long dwells under high force demonstrate that the motor stepping mechanism is inherently asymmetric, revealing an asymmetric phase in the kinesin walking cycle. Our findings support an asymmetric two-headed walking model for kinesin, with cooperative interactions between the two heads. The sensitivity of the 16-nm displacements to nucleotide and load raises the possibility that ADP release is a force-producing event of the kinesin cycle.
Higuchi
Hideo
H
Department of Metallurgy and Center for Interdisciplinary Research, Tohoku University, Sendai, Japan.
Bronner
Christian Eric
CE
Park
Hee-Won
HW
Endow
Sharyn A
SA
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D019281:Dimerization; D004331:Drosophila melanogaster / Q000235:genetics; D016547:Kinesins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D007834:Lasers; D008870:Microtubules / Q000378:metabolism*; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000378:metabolism; D009068:Movement; D009154:Mutation / Q000235:genetics*; D049329:Nanostructures; D020836:Protein Structure, Quaternary; D013912:Threonine / Q000235:genetics / Q000378:metabolism; D013997:Time Factors
D020409:Molecular Motor Proteins; D013912:Threonine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D016547:Kinesins