The interaction of the B-ring of colchicine with α-Tubulin: A novel footprinting approach

Asish R. Chaudhuri, P. Seetharamalu, Patricia M. Schwarz, F. H. Hausheer, Richard F. Ludueña

Research output: Contribution to journalArticle

Abstract

Tubulin, the major structural component of the microtubules, participates actively in mitotic spindle formation and chromosomal organization during cell division. Tubulin is the major target for a variety of anti-mitotic drugs. Some of the drugs, such as Vinca alkaloids and taxol, are routinely used for cancer chemotherapy. It is unfortunate that our knowledge of the binding sites on tubulin of these drugs is limited because of lack of a useful and appropriate tool. The photoaffinity labeling approach is the major technique available at present to detect the binding sites of drugs on tubulin. This method, however, has several limitations. First, only part of the binding site can be identified, namely, the residues which react with the photoaffinity label. Second, there are regions of tubulin which are not at the binding site but are affected by the binding of the drug; these regions can not be detected by the photoaffinity labeling approach. The third, and perhaps most serious, limitation is that the traditional approach can detect areas which have nothing to do with the binding of the ligand but which are within a certain distance of the binding site, that distance being less than the length of the photoreactive moiety attached to the ligand. There has been a great deal of controversy on the localization of the binding site of colchicine on tubulin, with some reports suggesting that the binding site is on α and some supporting a binding site on β. Colchicine also has significant effects on tubulin conformation, but the regions which are affected have not been identified. We have attempted here to address these questions by a novel 'footprinting' method by which the drug-binding sites and as well as the domain of tubulin affected by drug-induced conformational changes could be determined. Here, we report for the first time that the interaction of the B-ring of colchicine with the α-subunit affects a domain of tubulin which appears to be far from its binding site. This domain includes the cysteine residues at positions 295, 305, 315 and 316 on α-tubulin; these residues are located well away from the α/β interface where colchicine appears to bind. This is correlated with the stabilizing effect of colchicine on the tubulin molecule. Furthermore, we also found that the B-ring of colchicine plays a major role in the stability of tubulin while the A and the C-rings have little effect on it. Our results therefore, support a model whereby colchicine binds at the α/β interface of tubulin with the B-ring on the α-subunit and the A and the C-rings on the β-subunit. (C) 2000 Academic Press.

Original languageEnglish (US)
Pages (from-to)679-692
Number of pages14
JournalJournal of Molecular Biology
Volume303
Issue number5
DOIs
StatePublished - Nov 10 2000
Externally publishedYes

Fingerprint

Colchicine
Tubulin
Binding Sites
Pharmaceutical Preparations
Photoaffinity Labels
Vinca Alkaloids
Ligands
Spindle Apparatus
Paclitaxel
Microtubules
Cell Division
Cysteine

Keywords

  • Alkylation
  • Colchicine
  • Footprinting
  • Sulfhydryl groups
  • Tubulin

ASJC Scopus subject areas

  • Virology

Cite this

Chaudhuri, A. R., Seetharamalu, P., Schwarz, P. M., Hausheer, F. H., & Ludueña, R. F. (2000). The interaction of the B-ring of colchicine with α-Tubulin: A novel footprinting approach. Journal of Molecular Biology, 303(5), 679-692. https://doi.org/10.1006/jmbi.2000.4156

The interaction of the B-ring of colchicine with α-Tubulin : A novel footprinting approach. / Chaudhuri, Asish R.; Seetharamalu, P.; Schwarz, Patricia M.; Hausheer, F. H.; Ludueña, Richard F.

In: Journal of Molecular Biology, Vol. 303, No. 5, 10.11.2000, p. 679-692.

Research output: Contribution to journalArticle

Chaudhuri, AR, Seetharamalu, P, Schwarz, PM, Hausheer, FH & Ludueña, RF 2000, 'The interaction of the B-ring of colchicine with α-Tubulin: A novel footprinting approach', Journal of Molecular Biology, vol. 303, no. 5, pp. 679-692. https://doi.org/10.1006/jmbi.2000.4156
Chaudhuri, Asish R. ; Seetharamalu, P. ; Schwarz, Patricia M. ; Hausheer, F. H. ; Ludueña, Richard F. / The interaction of the B-ring of colchicine with α-Tubulin : A novel footprinting approach. In: Journal of Molecular Biology. 2000 ; Vol. 303, No. 5. pp. 679-692.
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