Abstract
Kinesin-1 is an ATP-driven, processive motor that transports cargo along microtubules in a tightly regulated stepping cycle. Efficient gating mechanisms ensure that the sequence of kinetic events proceeds in the proper order, generating a large number of successive reaction cycles. To study gating, we created two mutant constructs with extended neck-linkers and measured their properties using single-molecule optical trapping and ensemble fluorescence techniques. Owing to a reduction in the inter-head tension, the constructs access an otherwise rarely populated conformational state in which both motor heads remain bound to the microtubule. ATP-dependent, processive backstepping and futile hydrolysis were observed under moderate hindering loads. On the basis of measurements, we formulated a comprehensive model for kinesin motion that incorporates reaction pathways for both forward and backward stepping. In addition to inter-head tension, we found that neck-linker orientation is also responsible for ensuring gating in kinesin.
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Acknowledgements
S.M.B. acknowledges support from grant GM51453, and S.S.R. acknowledges support from grant AR048565 from the US National Institutes of Health.
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B.E.C., W.M.B.-P. and J.O.L.A. designed and carried out experiments, collected and analyzed data, and cowrote the paper. B.E.C. and J.O.L.A. carried out modeling and fits. S.M.B. and S.S.R. helped to design experiments and analyze results, and cowrote the paper.
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Clancy, B., Behnke-Parks, W., Andreasson, J. et al. A universal pathway for kinesin stepping. Nat Struct Mol Biol 18, 1020–1027 (2011). https://doi.org/10.1038/nsmb.2104
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DOI: https://doi.org/10.1038/nsmb.2104
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