Characterization of the Taxol binding site on the microtubule. Identification of Arg282 in β-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol

Srinivasa Rao, Lifeng He, Subrata Chakravarty, Iwao Ojima, George A. Orr, Susan Band Horwitz

Research output: Contribution to journalArticle

159 Citations (Scopus)

Abstract

Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically β-tubulin. Our previous studies have indicated that [3H]3'-(p- azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of β-tubulin (Rao, S., Krauss, N.E., Heerding, J.M., Swindell, C.S., Ringel, I., Orr, G.A., and Horwitz, S.B. (1994) J. Biol. Chem. 269, 3132-3134) and [3H]2-(m- azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217- 233 of β-tubulin (Rao, S., Orr, G.A., Chaudhary, A.G., Kingston, D.G.I., and Horwitz, S.B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [3H]7- (benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [3H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue crosslinks to Arg282 in β-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in β-tubulin as determined by photoaffinity labeling and electron crystallography.

Original languageEnglish (US)
Pages (from-to)37990-37994
Number of pages5
JournalJournal of Biological Chemistry
Volume274
Issue number53
DOIs
StatePublished - Dec 31 1999

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Tubulin
Paclitaxel
Microtubules
Binding Sites
Amino Acids
Crystallography
clostripain
Labeling
Electrons
Peptides
Molecular modeling
benzophenone
Protein Sequence Analysis
Guanosine Triphosphate
Polymerization
Dimers
Arginine
Digestion
Polymers

ASJC Scopus subject areas

  • Biochemistry

Cite this

Characterization of the Taxol binding site on the microtubule. Identification of Arg282 in β-tubulin as the site of photoincorporation of a 7-benzophenone analogue of Taxol. / Rao, Srinivasa; He, Lifeng; Chakravarty, Subrata; Ojima, Iwao; Orr, George A.; Band Horwitz, Susan.

In: Journal of Biological Chemistry, Vol. 274, No. 53, 31.12.1999, p. 37990-37994.

Research output: Contribution to journalArticle

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abstract = "Photoaffinity labeling methods have allowed a definition of the sites of interaction between Taxol and its cellular target, the microtubule, specifically β-tubulin. Our previous studies have indicated that [3H]3'-(p- azidobenzamido)Taxol photolabels the N-terminal 31 amino acids of β-tubulin (Rao, S., Krauss, N.E., Heerding, J.M., Swindell, C.S., Ringel, I., Orr, G.A., and Horwitz, S.B. (1994) J. Biol. Chem. 269, 3132-3134) and [3H]2-(m- azidobenzoyl)Taxol photolabels a peptide containing amino acid residues 217- 233 of β-tubulin (Rao, S., Orr, G.A., Chaudhary, A.G., Kingston, D.G.I., and Horwitz, S.B. (1995) J. Biol. Chem. 270, 20235-20238). The site of photoincorporation of a third photoaffinity analogue of Taxol, [3H]7- (benzoyldihydrocinnamoyl) Taxol, has been determined. This analogue stabilizes microtubules polymerized in the presence of GTP, but in contrast to Taxol, does not by itself enhance the polymerization of tubulin to its polymer form. CNBr digestion of [3H]7-(benzoyldihydrocinnamoyl)Taxol-labeled tubulin, with further arginine-specific cleavage by clostripain resulted in the isolation of a peptide containing amino acid residues 277-293. Amino acid sequence analysis indicated that the photoaffinity analogue crosslinks to Arg282 in β-tubulin. Advances made by electron crystallography in understanding the structure of the tubulin dimer have allowed us to visualize the three sites of photoincorporation by molecular modeling. There is good agreement between the binding site of Taxol in β-tubulin as determined by photoaffinity labeling and electron crystallography.",
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