The interaction of the B-ring of colchicine with alpha-tubulin: A novel footprinting approach

Tubulin, the major structural component of the microtubules, participates a ctively in mitotic spindle formation and chromosomal organization during ce ll division. Tubulin is the major target for a variety of anti-mitotic drug s. Some of the drugs, such as Vinca alkaloids and taxol, are routinely used for cancer chemotherapy. It is unfortunate that our knowledge of the bindi ng sites on tubulin of these drugs is limited because of lack of a useful a nd appropriate tool. The photoaffinity labeling approach is the major techn ique available at present to detect the binding sites of drugs on tubulin. This method, however, has several limitations. First, only part of the bind ing site can be identified, namely, the residues which react with the photo affinity label. Second, there are regions of tubulin which are not at the b inding site but are affected by the binding of the drug; these regions can not be detected by the photoaffinity labeling approach. The third, and perh aps most serious, limitation is that the traditional approach can detect ar eas which have nothing to do with the binding of the ligand but which are w ithin a certain distance of the binding site, that distance being less than the length of the photoreactive moiety attached to the Ligand. There has b een a great deal of controversy on the localization of the binding site of colchicine on tubulin, with some reports suggesting that the binding site i s on cc and some supporting a binding site on beta. Colchicine also has sig nificant effects on tubulin conformation, but the regions which are affecte d have not been identified. We have attempted here to address these questio ns by a novel "footprinting" method by which the drug-binding sites and as well as the domain of tubulin affected by drug-induced conformational chang es could be determined. Here, we report for the first time that the interac tion of the B-ring of colchicine with the alpha -subunit affects a domain o f tubulin which appears to be far from its binding site. This domain includ es the cysteine residues at positions 295, 305, 315 and 316 on alpha -tubul in; these residues are located well away from the alpha/beta interface wher e colchicine appears to bind. This is correlated with the stabilizing effec t of colchicine on the tubulin molecule. Furthermore, we also found that th e B-ring of colchicine plays a major role in the stability of tubulin while the A and the C-rings have little effect on it. Our results therefore, sup port a model whereby colchicine binds at the alpha/beta interface of tubuli n with the B-ring on the alpha -subunit and the A and the C-rings on the be ta -subunit. (C) 2000 Academic Press.