Characterization of the Taxol Binding Site on the Microtubule

• DOI: 10.1074/jbc.270.35.20235
• Publication Date: 2002-07-26T15:15:23Z
• Authors: Srinivasa Rao; George A. Orr; Ashok G. Chaudhary; David G.I. Kingston; Susan Band Horwitz
• Keywords: 0301 basic medicine; Azides; Binding Sites; Paclitaxel; Molecular Sequence Data; Brain; Affinity Labels; Tritium; Peptide Mapping; Peptide Fragments; 03 medical and health sciences; Tubulin; Microtubule Proteins; Animals; Cattle; Electrophoresis, Polyacrylamide Gel; Taxoids; Trypsin; Amino Acid Sequence; Cyanogen Bromide; Chromatography, High Pressure Liquid

Photoaffinity labeling methods are being used to define the molecular contacts between taxol and its target protein, tubulin. Our laboratory has demonstrated previously that [3H]3'-(p-azidobenzamido)taxol photolabels the N-terminal 31 amino acids of beta-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). The interaction of a second photoaffinity analogue of taxol, [3H]2-(m-azidobenzoyl)taxol, with tubulin has been investigated. This analogue specifically photolabels beta-tubulin and the photolabeling is completed by both taxol and unlabeled 2-(m-azidobenzoyl)-taxol indicating a common binding domain. To identify the site(s) of photoincorporation, [3H]2-(m-azidobenzoyl)taxol-photolabeled beta-tubulin was subjected to sequential cyanogen bromide and tryptic digestions. Radiolabeled peptides were purified by reverse phase high performance liquid chromatography, and amino acid sequencing studies identified a peptide containing amino acid residues 217-231 of beta-tubulin as the major photolabeled domain.