THERMODYNAMICS OF LIGAND-INDUCED ASSEMBLY OF TUBULIN

The equilibrium assembly of purified GDP-tubulin into microtubules ind uced by taxol and Taxotere has been studied as a function of solution variables, ligand, and nucleotide, in 10 mM sodium phosphate buffers. Assembly is coupled to the binding of one taxoid molecule per tubulin heterodimer, while binding to the unassembled protein is not detected within ligand solubility limits. Linked functions analysis has indicat ed that two Mg2+ and no more H+ ions are bound per tubulin-taxoid poly merized, and the heat capacity change is negligible within experimenta l error (determined by van't Hoff analysis and by differential scannin g calorimetry), in contrast with drug-free control microtubule assembl y and with the abnormal polymerization of the tubulin-colchicine compl ex. The apparent enthalpy change is ca. 240 kJ mol-1 (calorimetry), an d the process is entropy driven. The apparent standard free energy cha nge of taxoid-induced elongation at 2 mM free Mg2+, pH 6.1-6.7, and 37 -degrees-C is -29.5 +/- 0.4 (taxol) or -31.5 +/-0.4 kJ mol-1 (Taxotere ). This is independent of taxoid excess, which has indicated that the process measured corresponds to the elongation equilibrium of the full y liganded protein. Comparison to elongation in the absence of drug su ggests an apparent linkage free energy change of binding and polymeriz ation of -11.3 +/- 1.2 kJ mol-1. The taxoid-induced elongation of GTP- tubulin proceeds with an increment of apparent free energy change of - 2.5 +/- 0.4 kJ mol-1 over GDP-tubulin. It is proposed that the taxoid binding changes the conformation of GDP-tubulin from inactive to activ e, allowing productive binding and elongation at the microtubule end. Among several possible model mechanisms discussed, it is particularly attractive to think of taxoids as double-sided ligands, which bind to tubulin at the microtubule end and participate in a lateral contact in terface with the newly added tubulin molecule. In the kinetic pathway of assembly, these ligands should bind first to inactive Mg2+-induced linear GDP-tubulin oligomers and transform them into active bidimensio nal polymerization nuclei.