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Allosteric inhibition of kinesin-5 modulates its processive directional motility

Nature Chemical Biology volume 2, pages 480–485 (2006)Cite this article

Abstract

Small-molecule inhibitors of kinesin-5 (refs. 1–3), a protein essential for eukaryotic cell division4, represent alternatives to antimitotic agents that target tubulin5,6. While tubulin is needed for multiple intracellular processes, the known functions of kinesin-5 are limited to dividing cells, making it likely that kinesin-5 inhibitors would have fewer side effects than do tubulin-targeting drugs. Kinesin-5 inhibitors, such as monastrol1, act through poorly understood allosteric mechanisms, not competing with ATP binding7,8. Moreover, the microscopic mechanism of full-length kinesin-5 motility is not known. Here we characterize the motile properties and allosteric inhibition of Eg5, a vertebrate kinesin-5, using a GFP fusion protein in single-molecule fluorescence assays9. We find that Eg5 is a processive kinesin whose motility includes, in addition to ATP-dependent directional motion, a diffusive component not requiring ATP hydrolysis. Monastrol suppresses the directional processive motility of microtubule-bound Eg5. These data on Eg5's allosteric inhibition will impact these inhibitors' use as probes and development as chemotherapeutic agents.

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Figure 1: Recombinant Eg5-GFP is a homotetramer and can functionally replace endogenous Eg5.
Figure 2: Full-length tetrameric Eg5 is a processive kinesin.
Figure 3: Monastrol decreases the speed and enhances microtubule-bound diffusive motion of individual Eg5 molecules.
Figure 4: The microtubule-bound diffusive motion of individual Eg5 molecules does not need ATP hydrolysis.

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Acknowledgements

We thank G. Woehlke and U. Peters for Nkin-GFP and DHP2, respectively. Support was provided in part by a Research Grant from the Human Frontier Science Program (C.F.S. and T.M.K.). C.F.S. acknowledges the Foundation for Fundamental Research on Matter and the DFG Center Molecular Physiology of the Brain. L.C.K. and E.J.G.P. are grateful for a VIDI fellowship (E.J.G.P.) from the Research Council for Earth and Life Sciences (ALW), with financial aid from the Netherlands Organization for Scientific Research (NWO). T.M.K., B.H.K., J.H.K. are grateful to the US National Institutes of Health/National Institute of General Medical Sciences (GM65933) for support. B.H.K. is a Merck postdoctoral fellow.

Author information

Author notes

  1. Benjamin H Kwok and Lukas C Kapitein: These authors contributed equally to this work.

Authors and Affiliations

  1. Laboratory of Chemistry and Cell Biology, The Rockefeller University, 1230 York Avenue, New York, 10021, New York, USA

    Benjamin H Kwok, Jeffrey H Kim & Tarun M Kapoor

  2. Department of Physics and Astronomy and Laser Centre, Vrije Universiteit, De Boelelaan 1081, 1081, HV Amsterdam, The Netherlands

    Lukas C Kapitein, Erwin J G Peterman & Christoph F Schmidt

  3. III. Physikalisches Institut, Fakultät für Physik, Georg-August-Universität, Friedrich-Hund-Platz 1, 37077, Göttingen, Germany

    Christoph F Schmidt

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  1. Benjamin H Kwok
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Corresponding authors

Correspondence to Erwin J G Peterman or Tarun M Kapoor.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Purification of TEV protease-cleaved Eg5-GFP fusion protein. (PDF 308 kb)

Supplementary Fig. 2

Eg5-GFP can functionally replace endogenous Eg5 motor. (PDF 176 kb)

Supplementary Fig. 3

Position traces of microtubule-bound Eg5-GFP over time. (PDF 210 kb)

Supplementary Fig. 4

Chemical structure of DHP2, 2. (PDF 160 kb)

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Kwok, B., Kapitein, L., Kim, J. et al. Allosteric inhibition of kinesin-5 modulates its processive directional motility. Nat Chem Biol 2, 480–485 (2006). https://doi.org/10.1038/nchembio812

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