Effect of Tubulin Self-Association on GTP Hydrolysis and Nucleotide Exchange Reactions

Tubulin nucleation, microtubule (MT) assembly, stability, and dynamics depend on GTP hydrolysis and nucleotide exchange reactions. We investigated how the self-association of isolated tubulin dimers affects the rate of GTP hydrolysis and the equilibrium of nucleotide exchange. We used HPLC to determine the concentrations of GDP and GTP and thereby the GTPase activity of SEC-eluted tubulin dimers in assembly buffer solution, free of glycerol and tubulin aggregates. When GTP hydrolysis was negligible, the nucleotide exchange mechanism was studied using HPLC for determining the concentrations of tubulin-free and tubulin-bound GTP and GDP and by SAXS and cryo-TEM. We observed no GTP hydrolysis below the critical conditions for MT assembly, despite the assembly of tubulin 1D curved oligomers and single rings, showing that their assembly did not involve GTP hydrolysis under our conditions. Under conditions enabling spontaneous slow MT assembly, a slow pseudo-first-order GTP hydrolysis kinetics was detected, limited by the rate of MT assembly. Nucleotide exchange depended on the total tubulin concentration and the molar ratio between tubulin-free GDP and GTP. We used a thermodynamic model of isodesmic tubulin self-association, terminated by the formation of tubulin single-rings to calculate, at each tubulin concentration, the distributions of single rings, 1D oligomers, and free dimers, and thereby the molar fractions of dimers with exposed and buried nucleotide exchangeable sites (E-sites). Our analysis shows that the GDP to GTP exchange reaction equilibrium constant was an order-of-magnitude larger for tubulin dimers with exposed E-sites than for assembled dimers with buried E-sites.