Direct Photoaffinity Labeling by Dolastatin 10 of the Amino-terminal Peptide of β-Tubulin Containing Cysteine 12

Ruoli Bai, David G. Covell, George F. Taylor, John A. Kepler, Terry D. Copeland, Nga Y. Nguyen, George R. Pettit, Ernest Hamel
2004 Journal of Biological Chemistry  
Tubulin with bound [5-3 H]dolastatin 10 was exposed to ultraviolet light, and 8 -10% of the bound drug crosslinked to the protein, most of it specifically. The primary cross-link was to the peptide spanning amino acid residues 2-31 of ␤-tubulin, but the specific amino acid could not be identified. Indirect studies indicated that crosslink formation occurred between cysteine 12 and the thiazole moiety of dolastatin 10. An equipotent analog of dolastatin 10, lacking the thiazole ring, did not
more » ... e ring, did not form an ultraviolet light-induced cross-link to ␤-tubulin. Preillumination of tubulin with ultraviolet light, known to induce cross-link formation between cysteine 12 and exchangeable site nucleotide, inhibited the binding of [5-3 H]dolastatin 10 and cross-link formation more potently than it inhibited the binding of colchicine or vinblastine to tubulin. Conversely, binding of dolastatin 10 to tubulin inhibited formation of the cross-link between cysteine 12 and the exchangeable site nucleotide. Dithiothreitol inhibited formation of the ␤-tubulin/dolastatin 10 cross-link but not the ␤-tubulin/exchangeable site nucleotide cross-link. Modeling studies revealed a highly favored binding site for dolastatin 10 at the ؉ end of ␤-tubulin in proximity to the exchangeable site GDP. Computational docking of an energy-minimized dolastatin 10 conformation at this site placed the thiazole ring of dolastatin 10 8 -9 Å from the sulfur atom of cysteine 12. Dolastatin 15 and cryptophycin 1 could also be docked into positions that overlapped more extensively with the docked dolastatin 10 than with each other. This result was consistent with the observed binding properties of these peptides.
doi:10.1074/jbc.m402110200 pmid:15123603 fatcat:dxtomzoxzzcipaonblnhek34h4