Weak and strong states of kinesin and ncd.
Kinesin superfamily molecular motors step along microtubules (MTs) via a cycle of conformational changes which is coupled to ATP turnover. To probe the coupling mechanism, we titrated the effects of various nucleotides on MT binding by two superfamily members; MT plus-end-directed kinesin and MT minus-end-directed non claret disjunctional (ncd). For both motors, the nucleotide-free state induced by apyrase was the strongest binding (K(kin)d approximately 0.003 micro M, K(ncd)d approximately 0.24 micro M), whilst the ADp state was the weakest binding (K(kin)d approximately 11.32 micro M, K(ncd)d approximately 12.02 micro M). In ATP, the motor. ADP state dominates and the binding is accordingly ADP-like, but in the presence of the slowly hydrolysed analogue adenosine 5'-O-(3-thiotriphosphate) there is a shift towards tighter binding (K(kin)d approximately 4.23 micro M, K(ncd)d approximately 2.34 micro M), consistent with a tight-binding motor. ATP-like state being enriched. In the presence of non-hydrolysable analogue beta,gamma-imidoadenosine 5'-triphosphate the binding is still tighter (K(kin)d approximately <0.27 micro M, K(ncd)d approximately 0.21 micro M), close to the values obtained with apyrase. For both kinesin and ncd, ADP has the unique quality that it traps the motor in a weak binding state. MT tight binding catalyses escape from this state, changing the active site conformation such that both ADP release and ADP binding are accelerated. The data are consistent with a simple two-state scheme in which both kinesis and ncd switch from weak to strong binding via ADP release, and back again via ADP trapping. In a two-state model, the transition from weak to strong binding is force-generating.
Crevel
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IM
Molecular Motors Group Marie Curie Research Institute, Oxted, Surrey, UK.
Lockhart
A
A
Cross
R A
RA
D000818:Animals; D029721:Drosophila Proteins; D016547:Kinesins / Q000378:metabolism*; D008868:Microtubule Proteins / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D013552:Swine
D029721:Drosophila Proteins; D008868:Microtubule Proteins; D011994:Recombinant Proteins; C066696:ncd protein, Drosophila; D016547:Kinesins