Abstract
Kinesin-1 (conventional kinesin) is a dimeric motor protein that carries cellular cargoes along microtubules1,2 by hydrolysing ATP3 and moving processively in 8-nm steps4. The mechanism of processive motility involves the hand-over-hand motion of the two motor domains (‘heads’)5,6,7, a process driven by a conformational change in the neck-linker domain of kinesin8,9,10,11,12. However, the ‘waiting conformation’ of kinesin between steps remains controversial13,14,15,16—some models propose that kinesin adopts a one-head-bound intermediate17,18,19,20,21, whereas others suggest that both the kinesin heads are bound to adjacent tubulin subunits7,22,23. Addressing this question has proved challenging, in part because of a lack of tools to measure structural states of the kinesin dimer as it moves along a microtubule. Here we develop two different single-molecule fluorescence resonance energy transfer (smFRET) sensors to detect whether kinesin is bound to its microtubule track by one or two heads. Our FRET results indicate that, while moving in the presence of saturating ATP, kinesin spends most of its time bound to the microtubule with both heads. However, when nucleotide binding becomes rate-limiting at low ATP concentrations, kinesin waits for ATP in a one-head-bound state and makes brief transitions to a two-head-bound intermediate as it walks along the microtubule. On the basis of these results, we suggest a model for how transitions in the ATPase cycle position the two kinesin heads and drive their hand-over-hand motion.
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Acknowledgements
We thank M. Nakajima for support in cloning; H. Tadakuma and N. Stuurman for microscope construction and technical support; C. Shingyoji for the gift of sea urchin sperm; K. Kikuchi for the tracking program; and A. Yildiz and A. Carter for discussions. M.T. is supported by Grant-in-Aid for Scientific Research on Priority Areas from MEXT, Japan, a Research Grant for Young Investigators from the Human Frontier Science Program, and grants from the Mitsubishi Foundation and the Asahi Glass Foundation. R.D.V. is supported by grants from the Howard Hughes Medical Institute and the US National Institutes of Health.
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Mori, T., Vale, R. & Tomishige, M. How kinesin waits between steps. Nature 450, 750–754 (2007). https://doi.org/10.1038/nature06346
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DOI: https://doi.org/10.1038/nature06346
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