Conformational States of Ras Complexed with the GTP Analogue GppNHp or GppCH2p: Implications for the Interaction with Effector Proteins

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Abstract
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 31P NMR spectroscopy. Here we show that Ras(wt)·Mg2+·GppCH2p also exists in two conformational states in dynamic equilibrium. The activation enthalpy ΔH⧧12and the activation entropy ΔS⧧12for 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)·Mg2+·GppNHp complex. The same is true for the equilibrium constants K12 = [2]/[1] of 2.0 and the corresponding ΔG12of −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 31P−31P NOESY experiments which lead to a correction of the already reported assignments of bound GppNHp. Mutation of Thr35 in Ras·Mg2+·GppCH2p 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 31P 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 γ-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.