Conformational transitions in p21ras and in its complexes with the effector protein Raf-RBD and the GTPase activating protein GAP.
31P NMR revealed that the complex of p21ras with the GTP analog GppNHp.Mg2+ exists in two conformational states, states 1 and 2. In wild-type p21ras the equilibrium constant K1(12) between the two states is 1.09. The population of these states is different for various mutants but independent of temperature. The activation enthalpy delta H ++ and activation entropy delta S ++ for the conformational transitions were determined by full-exchange matrix analysis for wild-type p21ras and p21ras(S65P). For the wild-type protein one obtains delta H ++ = 89 +/- 2 kJ mol-1 and delta S ++ = 102 +/- 20 J mol-1 K-1 and for the mutant protein delta H ++ = 93 +/- 7 kJ mol-1 and delta S ++ = 138 +/- 30 J mol-1 K-1. The study of various p21ras mutants suggests that the two states correspond to different conformations of loop L2, with Tyr-32 in two different positions relative to the bound nucleotide. High-field EPR at 95 GHz suggest that the observed conformational transition does not directly influence the coordination sphere of the protein-bound metal ion. The influence of this transition on loop L4 was studied by 1H NMR with mutants E62H and E63H. There was no indication that L4 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 states is slow on the NMR time scale (< 10 s-1): at approximately pH 5 the population of the two states is equal. The interaction of p21ras-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 31P NMR spectroscopy. In complex with Raf-RBD the second conformation of p21ras (state 2) is stabilized. In this conformation Tyr-32 is located in close proximity to the phosphate groups of the nucleotide, and the beta-phosphate resonance is shifted upfield by 0.7 ppm. Spectra obtained in the presence of GAP suggest that in the ground state GAP does not interact directly with the nucleotide bound to p21ras and does not induce larger conformational changes in the neighborhood of the nucleotide. The experimental data are consistent with a picture where GAP accelerates the exchange process between the two states and simultaneously increases the population of state 1 at higher temperature.
Geyer
M
M
Max-Planck-Institut für medizinische Forschung, Berlin, Germany.
Schweins
T
T
Herrmann
C
C
Prisner
T
T
Wittinghofer
A
A
Kalbitzer
H R
HR
D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D020690:GTPase-Activating Proteins; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010759:Phosphorus Isotopes; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020703:ras GTPase-Activating Proteins
D020690:GTPase-Activating Proteins; D010759:Phosphorus Isotopes; D011506:Proteins; D011518:Proto-Oncogene Proteins; D020703:ras GTPase-Activating Proteins; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras)