Slow conformational dynamics of the guanine nucleotide-binding protein Ras complexed with the GTP analogue GTPγS

The guanine nucleotide-binding protein Ras occurs in solution in two different conformational states, state 1 and state 2 with an equilibrium constant K-12 of 2.0, when the GTP analogue guanosine-5'-(beta,gamma-imido) triphosphate or guanosine-5'-(b,c-methyleno) triphosphate is bound to the active centre. State 2 is assumed to represent a strong binding state for effectors with a conformation similar to that found for Ras complexed to effectors. In the other state ( state 1), the switch regions of Ras are most probably dynamically disordered. Ras variants that exist predominantly in state 1 show a drastically reduced affinity to effectors. In contrast, Ras(wt) bound to the GTP analogue guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S) leads to P-31 NMR spectra that indicate the prevalence of only one conformational state with K-12 > 10. Titration with the Ras-binding domain of Raf-kinase (Raf-RBD) shows that this state corresponds to effector binding state 2. In the GTP gamma S complex of the effector loop mutants Ras(T35S) and Ras(T35A) two conformational states different to state 2 are detected, which interconvert over a millisecond time scale. Binding studies with Raf-RBD suggest that both mutants exist mainly in low-affnity states 1a and 1b. From line-shape analysis of the spectra measured at various temperatures an activation energy Delta H-1a1b(vertical bar) of 61 kJ(.)mol(-1) and an activation entropy Delta S-1a1b(vertical bar) of 65 J(.)K(-1.)mol(-1) are derived. Isothermal titration calorimetry on Ras bound to the different GTP-analogues shows that the effective affinity K-A for the Raf-RBD to Ras(T35S) is reduced by a factor of about 20 compared to the wild-type with the strongest reduction observed for the GTP gamma S complex.