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Weak and Strong States of Kinesin and ncd

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Abstract

Kinesin superfamily molecular motors step along microtubules (MTs)viaa 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 (Kkind∼0.003 μM,Kncdd∼0.24 μM), whilst the ADP state was the weakest binding (Kkind∼11.32 μM,Kncdd∼12.02 μM). 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 (Kkind∼4.23 μM,Kncdd∼2.34 μM), consistent with a tight-binding motor.ATP-like state being enriched. In the presence of non-hydrolysable analogue β,γ- imidoadenosine 5′-triphosphate the binding is still tighter (Kkind∼<0.27 μM,Kncdd∼0.21 μM), 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 kinesin and ncd switch from weak to strong bindingviaADP release, and back againviaADP trapping. In a two-state model, the transition from weak to strong binding is force-generating.

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    Our new model (Fig. 6) is conventional in that ATP binding directly or indirectly gates forward stepping, with gating involving a combination of ATP-dependent unparking of the leading (tethered) head (4,5,35,36) and ATP-dependent stabilization of neck linker docking on the MT-attached trail head (37–40). Assigning K⋅ADP as the weakest binding state in the cycle is also conventional (27,28), and our assignment of the K⋅ADP-K⋅ADP state as the detaching state is also consistent with earlier models (10,15,41). The idea that some backward events are slips is also not novel; 16-, 24-nm, and larger backward displacements have previously been assigned as slips on the basis that they are too fast to represent a sequence of 8-nm steps that are each coupled to a full cycle of ATP turnover (15).

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