The B-ring substituent at C-7 of colchicine and the alpha-C-terminus of tubulin communicate through the "tail-body" interaction.

The carboxy terminals of alphabeta-tubulins are flexible regions rich in acidic amino acid residues that play an inhibitory role in the polymerization of tubulin to microtubules. We have shown that the binding of colchicine and its B-ring analogs (with C-7 substituents) to tubulin are pH sensitive and have high activation energies. Under identical conditions, the binding of analogs without C-7 substituents is pH independent and has lower activation energy. Beta-C-terminus-truncated tubulin (alphabeta(s)) shows similar pH sensitivity and activation energy to native tubulin (alphabeta). Removal of the C-termini of both subunits of tubulin (alpha(s)beta(s)) or the binding of a basic peptide P2 to the negatively charged alpha-C-terminus of tubulin causes a colchicine-tubulin interaction independent of pH with a low activation energy. Tubulin dimer structure shows that the C-terminal alpha-tail is too far from the colchicine binding site to interact directly with the bound colchicine. Therefore, it is likely that the interaction of the alpha-C-terminus with the main body of tubulin indirectly affects the colchicine-tubulin interaction via conformational changes in the main body. We therefore conclude that in the presence of tail-body interaction, a B-ring substituent makes contact with the alpha-tubulin and induces significant conformational changes in alpha-tubulin.

BisANS binding to tubulin: isothermal titration calorimetry and the site-specific proteolysis reveal the GTP-induced structural stability of tubulin.

Interactions of bisANS and ANS to tubulin in the presence and absence of GTP were investigated, and the binding and thermodynamic parameters were determined using isothermal titration calorimetry. Like bisANS binding to tubulin, we observed a large number of lower affinity ANS binding sites (N1 = 1.3, K1 = 3.7 x 10(5) M(-1), N2 = 10.5, K2 = 7 x 10(4)/M(-1)) in addition to 1-2 higher affinity sites. Although the presence of GTP lowers the bisANS binding to both higher and lower affinity sites (N1 = 4.3, N2 = 11.7 in absence and N1 = 1.8, N2 = 3.6 in presence of GTP), the stoichiometries of both higher and lower affinity sites of ANS remain unaffected in the presence of GTP. BisANS-induced structural changes on tubulin were studied using site-specific proteolysis with trypsin and chymotrypsin. Digestion of both alpha and beta tubulin with trypsin and chymotrypsin, respectively, has been found to be very specific in presence of GTP. GTP has dramatic effects on lowering the extent of nonspecific digestion of beta tubulin with trypsin and stabilizing the intermediate bands produced from both alpha and beta. BisANS-treated tubulin is more susceptible to both trypsin and chymotrypsin digestion. At higher bisANS concentration (>20 microM) both alpha and beta tubulins are almost totally digested with enzymes, indicating bisANS-induced unfolding or destabilization of tubulin structure. Again, the addition of GTP has remarkable effect on lowering the bisANS-induced enhanced digestion of tubulin as well as stabilizing effect on intermediate bands. These results of isothermal titration calorimetry, proteolysis and the DTNB-kinetics data clearly established that the addition of GTP makes tubulin compact and rigid and hence the GTP-induced stabilization of tubulin structure. No such destabilization of tubulin structure has been noticed with ANS, although, like bisANS, ANS possesses a large number of lower affinity binding sites. On the basis of these results, we propose that the unique structure of bisANS, which in absence of GTP can bind tubulin as a bifunctional ligand (through its two ANS moieties), is responsible for the structural changes of tubulin.