Controlling kinesin by reversible disulfide cross-linking: Identifying themotility-producing conformational change

Conventional kinesin, a dimeric molecular motor, uses ATP-dependent conform ational changes to move unidirectionally along a row of tubulin subunits on a microtubule. Two models have been advanced for the major structural chan ge underlying kinesin motility: the first involves an unzippering/zippering of a small peptide (neck linker) from the motor catalytic core and the sec ond proposes an unwinding/rewinding of the adjacent coiled-coil (neck coile d-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 crosslinking, kinesin ceased unidirectional moveme nt and only showed brief one-dimensional diffusion along microtubules. Moti lity fully recovered upon adding reducing agents to reverse the cross-link. When the neck linker motion was partially restrained, single kinesin motor s showed biased diffusion towards the microtubule plus end but could not mo ve effectively against a load imposed by an optical trap. Thus, partial mov ement of the neck linker suffices for directionality but not for normal pro cessivity or force generation. In contrast, preventing neck coiled-coil unw inding by disulfide cross-linking had relatively little effect on motor act ivity, although the average run length of single kinesin molecules decrease d by 30-50%. These studies indicate that conformational changes in the neck linker, not in the neck coiled-coil, drive processive movement by the kine sin motor.