TY - JOUR
T1 - 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
AU - Rao, Srinivasa
AU - He, Lifeng
AU - Chakravarty, Subrata
AU - Ojima, Iwao
AU - Orr, George A.
AU - Horwitz, Susan Band
PY - 1999/12/31
Y1 - 1999/12/31
N2 - 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.
AB - 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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U2 - 10.1074/jbc.274.53.37990
DO - 10.1074/jbc.274.53.37990
M3 - Article
C2 - 10608867
AN - SCOPUS:0033621479
SN - 0021-9258
VL - 274
SP - 37990
EP - 37994
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 53
ER -