LINKAGES BETWEEN THE DISSOCIATION OF ALPHA-BETA TUBULIN INTO SUBUNITSAND LIGAND-BINDING - THE GROUND-STATE OF TUBULIN IS THE GDP CONFORMATION

The effects of ligands on the dissociation of the alphabeta tubulin di mer into the two subunits were investigated using calf brain tubulin. Sedimentation equilibrium studies showed a number of linkages. In the absence of magnesium in the medium, tubulin-GTP, tubulin-GDP, and tubu lin with the exchangeable site unoccupied associate with essentially t he same strength (K(alphabeta) = 1 X 10(7) M-1). This indicates that t he ground state of tubulin (i.e., in the absence of magnesium) is not affected by occupancy of the exchangeable nucleotide binding site (E s ite). The alphabeta association is enhanced by magnesium ions. The ass ociation of tubulin with GDP in the E site is linked to the uptake of twice as many magnesium ions as that of tubulin with GTP in the E site . This suggests that magnesium binding is linked to an E-site-related conformational change. Consideration of the linkages between the bindi ng of magnesium ions, E-site occupancy, and tubulin conformation in te rms of the model [Howard, W. D., & Timasheff, S. N. (1986) Biochemistr y 25, 8292-8300] in which the tubulin alphabeta dimer exists in an equ ilibrium between two conformations, a microtubule-forming (''straight' ') state favored by GTP and a double-ring-forming (''curved'') state f avored by GDP, leads to the conclusion that the ground state of tubuli n is the ring-forming or ''curved'' conformation. Thus, in the absence of magnesium, the tubulin heterodimer exists in the ring-forming conf ormation, whether the E site is occupied by GTP or GDP. It is the stro ng binding (K(b) is similar to 8 X 10(5) M-1) of one magnesium ion to tubulin-GTP in the straight conformation that displaces the equilibriu m from the ring-forming state toward the microtubule-forming conformat ion. It is proposed that it is this linkage between a very unfavorable equilibrium (K is similar to 0.01) from the ring-forming, or storage, form of tubulin to the microtubule assembly-competent state with the binding of a single magnesium ion which is the control mechanism for t he activation of microtubule assembly and that this magnesium ion is l ikely the one complexed to the gamma-phosphate of GTP in the E site of tubulin. It is also this coupling of the strong binding of the magnes ium to the very weak conformational equilibrium that explains the requ irement of a high Mg2+ concentration in the microtubule assembly react ion. Dissociation of the alphabeta dimer was used to probe the nature of the nonexchangeable nucleotide site (N site). Incubation of tubulin , diluted to a level where alpha-beta dissociation occurs, with a 5000 -fold excess of GDP showed no evidence of nucleotide exchange at the N site, leading to the conclusion that GTP must occupy the N site with an affinity 10(6)-10(7) times greater than that for the E site.