Abstract
Kinesin undergoes a global folding conformational change from an extended active conformation at high ionic concentrations to a compact inhibited conformation at physiological ionic concentrations. Here we show that much of the observed ATPase activity of folded kinesin is due to contamination with proteolysis fragments that can still fold, but retain an activated ATPase function. In contrast, kinesin that contains an intact IAK-homology region exhibits pronounced inhibition of its ATPase activity (140-fold in 50 mM KCl) and weak net affinity for microtubules in the presence of ATP, resulting from selective inhibition of the release of ADP upon initial interaction with a microtubule. Subsequent processive cycling is only partially inhibited. Fusion proteins containing residues 883–937 of the kinesin α-chain bind tightly to microtubules; exposure of this microtubule-binding site in proteolysed species is probably responsible for their activated ATPase activities at low microtubule concentrations.
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References
Vale, R. D. & Fletterick, R. J. The design plan of kinesin motors. Annu. Rev. Cell Dev. Biol. 13, 745–777 (1997).
Hirokawa, N., Noda, Y. & Okada, Y. Kinesin and dynein superfamily proteins in organelle transport and cell division. Curr. Opin. Cell Biol. 10, 60–73 (1998).
Kirchner, J., Woehlke, G. & Schliwa, M. Universal and unique features of kinesin motors: insights from a comparison of fungal and animal conventional kinesins. Biol. Chem. 380, 915–921 (1999).
Diefenbach, R. J., Mackay, J. P., Armati, P. J. & Cunningham, A. L. The C-terminal region of the stalk domain of ubiquitous human kinesin heavy chain contains the binding site for kinesin light chain. Biochemistry 37, 16663–16670 (1998).
Verhey, K. J. et al. Light chain-dependent regulation of kinesin’s interaction with microtubules. J. Cell Biol. 143, 1053–1066 (1998).
Howard, J., Hudspeth, A. J. & Vale, R. D. Movement of microtubules by single kinesin molecules. Nature 342, 154–158 (1989).
Block, S. M., Goldstein, L. S. B. & Schnapp, B. J. Bead movement by single kinesin molecules studied with optical tweezers. Nature 348, 348–352 (1990).
Hackney, D. D. Highly processive microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains. Nature 377, 448–450 (1995).
Hackney, D. D. Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. Proc. Natl Acad. Sci. USA 91, 6865–6869 (1994).
Rice, S. et al. A structural change in the kinesin motor protein that drives motility. Nature 402, 778–784 (1999).
Jiang, W., Stock, M., Li, X. & Hackney, D. D. Influence of the kinesin neck domain on dimerization and ATPase kinetics. J. Biol. Chem. 272, 7626–7632 (1997).
Wagner, M. C., Pfister, K. K., Bloom, G. S. & Brady, S. T. Copurification of kinesin polypeptides with microtubule- stimulated magnesium ATPase activity and kinetic analysis of enzymic properties. Cell Motil. Cytoskeleton 12, 195–215 (1989).
Hackney, D. D., Levitt, J. D. & Wagner, D. D. Characterization of α2β2 and α2 forms of kinesin. Biochem. Biophys. Res. Comm. 174, 810–815 (1991).
Jiang, M. Y. & Sheetz, M. P. Cargo-activated ATPAse activity of kinesin. Biophys. J. 68 (Suppl.), 283–285 (1995).
Moraga, D. E. & Murphy, D. B. Kinesin is ‘inactive’ unless bound to a solid support. Mol. Biol. Cell 8, 258a–258a (1997).
Coy, D. L., Hancock, W. O., Wagenbach, M. & Howard, J. Kinesin’s tail domain is an inhibitory regulator of the motor domain. Nature Cell Biol. 1, 288–292 (1999).
Hisanaga, S. et al. The molecular structure of adrenal medulla kinesin. Cell Motil. Cytoskeleton 12, 264–272 (1989).
Hackney, D. D., Levitt, J. D. & Suhan, J. Kinesin undergoes a 9S to 6S conformational transition. J. Biol. Chem. 267, 8696–8701 (1992).
Stock, M. F. et al. Formation of the compact conformer of kinesin requires a C-terminal heavy chain domain and inhibits microtubule-stimulated ATPase activity. J. Biol. Chem. 274, 14617–14623 (1999).
Friedman, D. S. & Vale, R. D. Single-molecule analysis of kinesin motility reveals regulation by the cargo-binding tail domain. Nature Cell Biol. 1, 293–297 (1999).
Navone, F. et al. Cloning and expression of a human kinesin heavy chain gene: interaction of the COOH-terminal domain with cytoplasmic microtubules in transfected CV-1 cells. J. Cell Biol. 117, 1263–1275 (1992).
Cross, R. A., Jackson, A. P., Citi, S., Kendrick-Jones, J. & Bagshaw, C. R. Active site trapping of nucleotide by smooth and non-muscle myosins. J. Mol. Biol. 203, 173–181 (1988).
Cheng, J. Q., Jiang, W. & Hackney, D. D. Interaction of mant-adenosine nucleotides and magnesium with kinesin. Biochemistry 37, 5288–5295 (1998).
Hackney, D. D. The rate limiting step in microtubule-stimulated ATP hydrolysis by dimeric kinesin head domains occurs while bound to the microtubule. J. Biol. Chem. 269, 16508–16511 (1994).
Kirchner, J., Seiler, S., Fuchs, S. & Schliwa, M. Functional anatomy of the kinesin molecule in vivo. EMBO J. 18, 4404–4413 (1999).
Hackney, D. D. Isolation of kinesin using initial batch ion exchange. Methods Enzymol. 196, 175–181 (1991).
Huang, T.-G. & Hackney, D. D. Drosophila kinesin minimal motor domain expressed in Escherichia coli. Purification and kinetic characterization. J. Biol. Chem. 269, 16493–16501 (1994).
Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254 (1976).
Neuhoff, V., Arold, N., Taube, D. & Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brillant Blue G-250 and R-250. Electrophoresis 9, 255–262 (1988).
Ingold, A. L., Cohn, S. A. & Scholey, J. M. Inhibition of kinesin-driven microtubule motility by monoclonal antibodies to kinesin heavy chains. J. Cell Biol. 107, 2659–2670 (1988).
Acknowledgements
We thank J. Scholey for hybridoma cells expressing the SUK4 antibody. This work was supported by NIH grant NS28562.
Correspondence and requests for materials should be addressed to D.D.H.
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Hackney, D., Stock, M. Kinesin’s IAK tail domain inhibits initial microtubule-stimulated ADP release. Nat Cell Biol 2, 257–260 (2000). https://doi.org/10.1038/35010525
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DOI: https://doi.org/10.1038/35010525
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