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© The Rockefeller University Press,
0021-9525/2000//1081 $5.00
The Journal of Cell Biology, Volume 151, Number 5,
, 2000 1081-1092
Original Article |
Controlling Kinesin by Reversible Disulfide Cross-Linking
: Identifying the Motility-Producing Conformational Change
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, 513 Parnassus Ave., San Francisco, CA 94143. Ph.: (415) 476-6380.(415) 502-1391
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.
Key Words: kinesin • processivity • disulfide cross-linking • neck linker • one-dimensional diffusion
© 2000 The Rockefeller University Press
The online version of this article contains supplemental material.Abbreviations used in this paper: a.a., amino acid; AMPPNP, 5'-adenylylimidodiphosphate; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); GFP, green fluorescent protein; MSD, mean-square-displacement; NEM, N-ethylmaleimide; oPDM, N,N'-1,2-phenylenedimaleimide; pPDM, N,N'-1,4-phenylenedimaleimide.
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