Controlling kinesin by reversible disulfide cross-linking. Identifying the motility-producing conformational change.
Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conformational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural change underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the second proposes an unwinding/rewinding of the adjacent coiled-coil (neck coiled-coil). Here, we have tested these models using disulfide cross-linking of cysteines engineered into recombinant kinesin motors. When the neck linker motion was prevented by cross-linking, kinesin ceased unidirectional movement and only showed brief one-dimensional diffusion along microtubules. Motility fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motors showed biased diffusion towards the microtubule plus end but could not move effectively against a load imposed by an optical trap. Thus, partial movement of the neck linker suffices for directionality but not for normal processivity or force generation. In contrast, preventing neck coiled-coil unwinding by disulfide cross-linking had relatively little effect on motor activity, although the average run length of single kinesin molecules decreased by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kinesin motor.
Tomishige
M
M
The Howard Hughes Medical Institute and the Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143, USA.
Vale
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RD
D003432:Cross-Linking Reagents / Q000737:chemistry / Q000378:metabolism; D004058:Diffusion; D019281:Dimerization; D004220:Disulfides / Q000378:metabolism*; D006801:Humans; D016547:Kinesins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism; D020409:Molecular Motor Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis / Q000502:physiology; D017433:Protein Structure, Secondary / Q000502:physiology
D003432:Cross-Linking Reagents; D004220:Disulfides; D020409:Molecular Motor Proteins; D016547:Kinesins