Effect of colchicine analogues 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 alpha beta tubulin association, nucleotide binding, and the interaction of colchicine 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 (alpha beta) tubulin and to one of the monomeric subunits, and the binding affinity for the dissociated 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 protein. For tubulin-GDP, the binding of the drug to the dissociated state was significantly weaker (6.3 x 10(4) M-1) than to the dimeric state, suggesting the existence of an E-site-related conformational change in the dissociated state. Podophyllotoxin, which contains the A-ring portion of colchicine, bound with equal affinity to the dimeric and dissociated 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-related conformational change.(ABSTRACT TRUNCATED AT 250 WORDS)