EFFECT OF COLCHICINE ANALOGS ON THE DISSOCIATION OF ALPHA-BETA-TUBULIN INTO SUBUNITS - THE LOCUS OF COLCHICINE BINDING

A combination of ligand binding and sedimentation equilibrium studies was used to characterize the thermodynamic linkages between alphabeta tubulin association, nucleotide binding, and the interaction of colchi cine analogues with dimeric and dissociated tubulins. The strength of binding of allocolchicine to the tubulin dimer was identical (8 x 10(5 ) M-1) whether the exchangeable nucleotide site (E site) was occupied by GTP or GDP. This drug bound to dimeric (alphabeta) tubulin and to o ne of the monomeric subunits, and the binding affinity for the dissoci ated state was linked to occupancy of the exchangeable nucleotide site . When the exchangeable site was occupied by GTP, the drug bound with very similar affinities to the dimeric and dissociated states of the p rotein. For tubulin-GDP, the binding of the drug to the dissociated st ate was significantly weaker (6.3 x 10(4) M-1) than to the dimeric sta te, suggesting the existence of an E-site-related conformational chang e in the dissociated state: Podophyllotoxin, which contains the A-ring portion of colchicine, bound with equal affinity to the dimeric and d issociated forms of both tubulin-GTP and tubulin-GDP, indicating that it is the C-ring portion of colchicine that is linked to the E-site-re lated conformational change. Given that the nonexchangeable nucleotide site does not exchange with free nucleotide following dimer dissociat ion [Shearwin, K. E., Perez-Ramirez, B., & Timasheff, S. N. (1994) (pr eceding paper in this issue)], the colchicine binding site and the exc hangeable site must be located on the same subunit; this is the beta s ubunit [Geahlen, R. L., & Haley, B. E. (1977) Proc. Natl. Acad. Sci. U .S.A. 74, 4375-4377]. Examination of the free energy linkages between drug binding and tubulin dimer dissociation shows that the alpha subun it can contribute at most 10% of the free energy of binding of the dru g to the dimer. It is proposed that the positioning of colchicine on t he beta subunit of tubulin is such that ring A is juxtaposed to the al pha-beta subunit interface.