A thermodynamic study of the interaction of tubulin with colchicine site ligands.

The bicyclic colchicine analogue 2-methoxy-5-(2',3',4'-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-on e (MTC) has been used to study the thermodynamics of specific ligand binding to the colchicine site of tubulin, employing isothermal reaction microcalorimetry. The binding of MTC to purified calf brain tubulin, in 10 mM sodium phosphate buffer, pH 7.0, is characterized by delta H degree = -19 +/- 1 kJ.mol-1, delta G degree = -31.8 +/- 0.6 kJ.mol-1, and delta S degree = 43 +/- 5 J.mol-1.K-1 at 298 K, with a slight variation in the temperature range from 283 to 308 K. The binding thermodynamics of colchicine and allocolchicine are similar to MTC under the conditions examined, suggesting related molecular interactions of the three ligands with the protein binding site. The standard enthalpy changes of binding of colchicine and MTC at 308 K coincide within experimental error. Therefore the more favorable free energy change of binding of colchicine must come from a larger binding entropy change (by about 20 J.mol-1.K-1). This difference could be attributed to the presence of the middle ring of colchicine, which is absent in MTC. Consistently, a similar entropy change is observed by the comparison of allocolchicine to MTC binding at several temperatures. In addition, allocolchicine binding is about 6 kJ.mol-1 less exothermic than MTC binding, which could be attributed to the presence in allocolchicine of a substituted phenyl ring instead of the colchicine-MTC tropolone ring. The present results and analysis are fully compatible with the previously proposed bifunctional binding of colchicine and MTC (through their trimethoxybenzene and tropolone moieties) to a bifocal protein binding site, and also with a partial immobilization of intramolecular rotation of MTC upon binding, which in colchicine is already constrained by its middle ring (Andreu, J. M., Gorbunoff, M. J., Lee, J. C., and Timasheff, S. (1984) Biochemistry 23, 1742-1752).