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A metal switch for controlling the activity of molecular motor proteins

Nature Structural & Molecular Biology volume 19, pages 122–127 (2012)Cite this article

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Abstract

Kinesins are molecular motors that require a divalent metal ion (for example, Mg2+) to convert the energy of ATP hydrolysis into directed force production along microtubules. Here we present the crystal structure of a recombinant kinesin motor domain bound to Mn2+ and ADP and report on a serine-to-cysteine substitution in the switch 1 motif of kinesin that allows its ATP hydrolysis activity to be controlled by adjusting the ratio of Mn2+ to Mg2+. This mutant kinesin binds ATP similarly in the presence of either metal ion, but its ATP hydrolysis activity is greatly diminished in the presence of Mg2+. In human kinesin-1 and kinesin-5 as well as Drosophila melanogaster kinesin-10 and kinesin-14, this defect is rescued by Mn2+, providing a way to control both the enzymatic activity and force-generating ability of these nanomachines.

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Figure 1: Crystal structure of Kin10–MnADP and the ATP hydrolysis–competent state for kinesin.
Figure 2: Metal and nucleotide binding to kinesin.
Figure 3: Mn2+ rescue of kinesin (serine-to-cysteine mutant) ATPase in the absence and presence of microtubules.
Figure 4: Acid-quench and phosphate release kinetics.
Figure 5: Mn2+ rescue of in vitro microtubule gliding.

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Acknowledgements

We thank V. Stojanoff of the National Synchrotron Light Source beam line X6A for help with data collection, R. Sloboda, A. Lavanway and H. Sardar for help with the fluorescence microscopy, and W. Casey for electron paramagnetic resonance training. The project was supported by F32AR054653 (US National Institutes of Health, National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH, NIAMS)) to J.C.C. and GM097079 (NIH, National Institute of General Medical Sciences (NIGMS)) to F.J.K., and research carried out at the X6A beam line was funded by the NIH, NIGMS, under agreement GM-0080. The National Synchrotron Light Source at Brookhaven National Laboratory is supported by the US Department of Energy, under contract DE-AC02-98CH10886.

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  1. Jared C Cochran

    Present address: Current address: Indiana University, Department of Molecular and Cellular Biochemistry, Bloomington, Indiana, USA.,

Authors and Affiliations

  1. Department of Chemistry, Dartmouth College, Hanover, New Hampshire, USA.,

    Jared C Cochran, Yu Cheng Zhao, Dean E Wilcox & F Jon Kull

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Contributions

J.C.C. cloned constructs, purified protein, crystallized, collected, processed and refined X-ray data with help from Y.C.Z. J.C.C. carried out kinetic assays, analyzed kinetic data, did EPR experiments, conducted microtubule gliding assays, and wrote the paper. D.E.W. and F.J.K. were involved in study conception, study design and editing the final manuscript. All authors discussed the results and commented on the manuscript.

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Correspondence to F Jon Kull.

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Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 1–2 and Supplementary Methods (PDF 478 kb)

Supplementary Movie 1

MT gliding by Kin1(WT) and Kin1(SC) areshown in the presence of MgATP and MnATP (MOV 2351 kb)

Supplementary Movie 2

Long time-lapse movie of MT gliding by Kin1(SC) in the presence of MgATP. (MOV 3430 kb)

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Cochran, J., Zhao, Y., Wilcox, D. et al. A metal switch for controlling the activity of molecular motor proteins. Nat Struct Mol Biol 19, 122–127 (2012). https://doi.org/10.1038/nsmb.2190

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