CONFORMATIONAL TRANSITIONS IN P21(RAS) AND IN ITS COMPLEXES WITH THE EFFECTOR PROTEIN RAF-RBD AND THE GTPASE-ACTIVATING PROTEIN GAP

P-31 NMR revealed that the complex of p21(ras) with the GTP analog Gpp NHp . Mg2+ exists in two conformational states, states 1 and 2. In wil d-type p21(ras) the equilibrium constant K-1((12)) between the two sta les is 1.09. The population of these stales is different for various m utants but independent of temperature. The activation enthalpy Delta H -double dagger and activation entropy Delta S-double dagger for the co nformational transitions were determined by full-exchange matrix analy sis for wild-type p21(ras) and p21(ras)(S65P). For the wildtype protei n one obtains Delta K-double dagger = 89 +/- 2 kJ mol(-1) and Delta S- double dagger = 102 +/- 20 J mol(-1) K-1 and for the mutant protein De lta K-double dagger = 93 +/- 7 kJ mol(-1) and Delta S-double dagger = 138 +/- 30 J mol(-1) K-1. The study of various p21(ras) mutants sugges ts that the two states correspond to different conformations of loop L 2, with Tyr-32 in two different positions relative to the bound nucleo tide. High-field EPR at 95 GHz suggests that the observed conformation al transition does not directly influence the coordination sphere of t he protein-bound metal ion. The influence of this transition on loop L 4 was studied by H-1 NMR with mutants E62H and E63H. There was no indi cation that LA takes part in the transition described in L2. although a reversible conformational change could be induced by decreasing the pH value. The exchange between the two slates is slow On the NMR time scale (<10 s(-1)), at approximately pH 5 the population of the two sta tes is equal. The interaction of p21(ras)-triphosphate complexes with the Ras-binding domain (RBD) of the effector protein c-Raf-1, Raf-RBD, and with the GTPase activating protein GAP was studied by P-31 NMR sp ectroscopy. In complex with Raf-RBD the second conformation of p21(ras ) (state 2) is stabilized. In this conformation Tyr-32 is located in c lose proximity to the phosphate groups of the nucleotide, and the beta -phosphate resonance is shifted upfield by 0.7 ppm. Spectra obtained I n the presence of GAP suggest that in the ground slate GAP does not in teract directly with the nucleotide bound to p21(ras) and does not ind uce larger conformational changes in the neighborhood of the nucleotid e. The experimental data are consistent with a picture where GAP accel erates the exchange process between the two states and simultaneously increases the population of slate 1 at higher temperature.