Mapping the binding site of colchicinoids on beta-tubulin: 2-chloroacetyl-2-demethylthiocolchicine covalently reacts predominantly with cysteine 239 and secondarily with cysteine 354

SUMMARY The colchicine analogs 2-chloroacetyl-2-demethylthiocolchicine (2CTC) and 3-chloroacetyl-3-demethylthiocolchicine (3CTC) are very similar to colchicine in their binding interaction with tubulin. Moreover, both analogs react covalently with the protein, forming adducts with cysteine residues 239 and 354 of b-tubulin. The adducts at Cys-239 appear to be less stable than those at Cys-354 in formic acid (used to generate peptides for sequence analysis). Extrapolating to zero time, we estimate that the Cys-239 to Cys-354 adduct ratio is 77:23 for 2CTC and 27:73 for 3CTC. When we used energy minimization modeling to dock colchicine, thiocolchicine, 2CTC, and 3CTC into a possible colchicine binding site in the electron crystallographic model of b-tubulin derived from docetaxel-stabilized protofilaments [Nogales et al. (1998) Nature (London) 391, 199-203], we found two potential binding sites. At one, entirely encompassed within the b-tubulin subunit, the C2- and C3-oxygen atoms of 2CTC and 3CTC did not overlap well with those of colchicine and thiocolchicine, but in this site the altered positions of the oxygen atoms in 2CTC and 3CTC resulted in qualitatively, but not quantitatively, consistent distances from the reactive carbon atoms of the analogs to the sulfur atoms of the cysteine residues. The other potential binding site was located at the a/b interface. With binding in this site, there was good overlap of the oxygen atoms of the analogs with those of colchicine, but relative distances of the reactive carbons in the analogs to the cysteine sulfur atoms did not correlate well with the observed reactivity. We conclude that a significant conformational change must occur in the colchicine binding site in the transition from the unpolymerized to the polymerized state of tubulin.

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