Identification of cysteine 354 of beta-tubulin as part of the binding site for the A ring of colchicine

The colchicine analog 3-chloroacetyl-3-demethylthiocolchicine (3CTC) is a competitive inhibitor of colchicine binding to tubulin, binds to tubulin at 37 degrees C, but not at 0 degrees C, and covalently reacts with beta-tubulin at 37 degrees C, but not at 0 degrees C, in a reaction inhibited by colchicine site drugs. The approximate intramolecular distance be tween the oxygen at position C-3 in 3CTC and the chlorine atom of the 3-chloroacetyl group is 3 Angstrom. Using decylagarose chromatography, we purified beta-tubulin that had reacted with 3-(chloromethyl-[C-14]carbonyl)-3-demethylthiocolchicine ([C-14]3CTC). This beta-tubulin was digested with formic acid, cyanogen bromide, endoproteinase Glu-C, or endoproteinase Lys C, and the radiolabeled peptide(s) were isolated. The sequences of these peptides indicated that as much as 90% of the covalent reaction between the [C-14]3CTC and beta-tubulin occurred at cysteine 354. This finding indicates that the C-3 oxygen atom of colchicinoids is within 3 Angstrom of the sulfur atom of the Cys-354 residue, suggests that the colchicine A ring lies between Cys-354 and Cys-239, based on the known 9 Angstrom distance between these residues, and may indicate that the tropolone C ring lies between the peptide region containing Cys-239 and the amino-terminal beta-tubulin sequence, based on the labeling pattern observed following direct photoactivation of tubulin-bound colchicine.