Abstract
Tubulin, the microtubule building-block, is the target of numerous small molecule compounds that interfere with microtubule dynamics. Several of these ligands are in clinical use as antitumor drugs. There have been numerous studies on these molecules, with two main objectives: to determine their mechanism of action and to find new compounds that would expand the arsenal available for cancer chemotherapy. Although these studies would undoubtedly benefit from structural data on tubulin, this protein has long resisted crystallization attempts. We have used stathmin-like domains (SLDs) of stathmin family proteins as a tool to crystallize tubulin and have obtained three-dimensional crystals of the tubulin:SLD complexes. As many tubulin ligands bind to these complexes, the crystals are valuable tools to study tubulin-drug interactions by X-ray crystallography. They open the way to a structure-based drug design approach.
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References
Schönbrunn, E., Phlippen, W., Trinczek, B., et al. (1999) Crystallization of a macromolecular ring assembly of tubulin liganded with the anti-mitotic drug podophyllotoxin. J. Struct. Biol. 128, 211–215.
Nogales, E., Wolf, S. G., and Downing, K. H. (1998) Structure of the ß-tubulin dimer by electron crystallography. Nature 391, 199–203.
Nettles, J. H., Li, H., Cornett, B., Krahn, J. M., Snyder, J. P., and Downing, K. H. (2004) The binding mode of epothilone A on ß-tubulin by electron crystallography. Science 305, 866–869.
Jourdain, L., Curmi, P., Sobel, A., Pantaloni, D., and Carlier, M.-F. (1997) Stathmin: a tubulin sequestering protein which forms a ternary T2S complex with two tubulin molecules. Biochemistry 36, 10,817–10,821.
Charbaut, E., Curmi, P. A., Ozon, S., Lachkar, S., Redeker, V., and Sobel, A. (2001) Stathmin family proteins display specific molecular and tubulin binding properties. J. Biol. Chem. 276, 16,146–16,154.
Gigant, B., Curmi, P. A., Martin-Barbey, C., et al. (2000) The 4 Å X-ray structure of a tubulin:stathmin-like domain complex. Cell 102, 809–816.
Ravelli, R. B., Gigant, B., Curmi, P. A., et al. (2004) Insight into tubulin regulation from a complex with colchicine and a stathmin-like domain. Nature 428, 198–202.
Gigant, B., Wang, C., Ravelli, R. B., et al. (2005) Structural basis for the regulation of tubulin by vinblastine. Nature 435, 519–522.
Williams, R. C., Jr. and Lee, J. C. (1982) Preparation of tubulin from brain, in Structural and Contractile Proteins Part B, (Frederiksen, D. and Cunningham, L., eds.), Academic Press, New York, pp. 376–385.
Castoldi, M. and Popov, A. V. (2003) Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr. Purif. 32, 83–88.
Lusty, C. J. (1999) A gentle vapor-diffusion technique for cross-linking of protein crystals for cryocrystallography. J. Appl. Cryst. 32, 106–112.
Rodgers, D. W. (1997) Practical cryocrystallography, in Macromolecular Crystallography Part A, (Carter, C. and Sweet, R., eds.), Academic Press, New York, pp. 183–202.
Hamel, E. and Lin, C. M. (1981) Glutamate-induced polymerization of tubulin: characteristics of the reaction and application to the large-scale purification of tubulin. Arch. Biochem. Biophys. 209, 29–40.
Vandecandelaere, A., Martin, S. R., Schilstra, M. J., and Bayley, P. M. (1994) Effects of the tubulin-colchicine complex on microtubule dynamic instability. Biochemistry 33, 2792–2801.
Andreu, J. M. and Timasheff, S. N. (1982) Conformational states of tubulin liganded to colchicine, tropolone methyl ether and podophyllotoxin. Biochemistry 21, 6465–6476.
Stura, E. A. (1999) Seeding techniques, in Crystallization of Nucleic Acids and Proteins, 2nd ed. (Ducruix, A. and Giege, R., eds.), University Press, Oxford, UK, pp. 177–208.
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Dorleans, A., Knossow, M., Gigant, B. (2007). Studying Drug-Tubulin Interactions by X-Ray Crystallography. In: Zhou, J. (eds) Microtubule Protocols. Methods in Molecular Medicine™, vol 137. Humana Press. https://doi.org/10.1007/978-1-59745-442-1_16
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DOI: https://doi.org/10.1007/978-1-59745-442-1_16
Publisher Name: Humana Press
Print ISBN: 978-1-58829-642-9
Online ISBN: 978-1-59745-442-1
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