Conformational states of Ras complexed with the GTP analogue GppNHp or GppCH2p: implications for the interaction with effector proteins.
The guanine nucleotide-binding protein Ras occurs in solution in two different states, state 1 and state 2, when the GTP analogue GppNHp is bound to the active center as detected by (31)P NMR spectroscopy. Here we show that Ras(wt).Mg(2+).GppCH(2)p also exists in two conformational states in dynamic equilibrium. The activation enthalpy DeltaH(++)(12) and the activation entropy DeltaS(++)(12) for the transition from state 1 to state 2 are 70 kJ mol(-1) and 102 J mol(-1) K(-1), within the limits of error identical to those determined for the Ras(wt).Mg(2+).GppNHp complex. The same is true for the equilibrium constants K(12) = [2]/[1] of 2.0 and the corresponding DeltaG(12) of -1.7 kJ mol(-1) at 278 K. This excludes a suggested specific effect of the NH group of GppNHp on the equilibrium. The assignment of the phosphorus resonance lines of the bound analogues has been done by two-dimensional (31)P-(31)P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras.Mg(2+).GppCH(2)p to serine leads to a shift of the conformational equilibrium toward state 1. Interaction of the Ras binding domain (RBD) of Raf kinase or RalGDS with Ras(wt) or Ras(T35S) shifts the equilibrium completely to state 2. The (31)P NMR experiments suggest that, besides the type of the side chain of residue 35, a main contribution to the conformational equilibrium in Ras complexes with GTP and GTP analogues is the effective acidity of the gamma-phosphate group of the bound nucleotide. A reaction scheme for the Ras-effector interaction is presented which includes the existence of two conformations of the effector loop and a weak binding state.
Spoerner
Michael
M
Institut für Biophysik und physikalische Biochemie, Universität Regensburg, Universitätsstrasse 31, 93053 Regensburg, Germany.
Nuehs
Andrea
A
Ganser
Petra
P
Herrmann
Christian
C
Wittinghofer
Alfred
A
Kalbitzer
Hans Robert
HR
D019943:Amino Acid Substitution / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D041961:Deuterium Exchange Measurement; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010710:Phosphates / Q000737:chemistry / Q000378:metabolism; D010759:Phosphorus Isotopes / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D019908:Proto-Oncogene Proteins c-raf / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D020726:ral Guanine Nucleotide Exchange Factor / Q000737:chemistry / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
D002413:Cations, Divalent; D010710:Phosphates; D010759:Phosphorus Isotopes; D020726:ral Guanine Nucleotide Exchange Factor; D006165:Guanylyl Imidodiphosphate; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate; D019908:Proto-Oncogene Proteins c-raf; D018631:ras Proteins; D008274:Magnesium