Conformational states of the nuclear GTP-binding protein Ran and its complexes with the exchange factor RCC1 and the effector protein RanBP1

It has been shown before by P-31 NMR that Ras bound to the nonhydrolyzable GTP analogue guanosine 5'-O-(beta,gamma-imidotriphosphate) (GppNHp) exists in two conformations which are rapidly interconverting with a rate constant of 3200 s(-1) at 30 degrees C [Geyer, M., et al. (1996) Biochemistry 35, 1 0308-19320]. Here we show that Ran complexed with GTP also exists in two co nformational states, 1 and 2, which can be directly inferred from the occur rence of two P-31 NMR resonance lines for the gamma-phosphate group of boun d GTP. The exchange between the two states is slow on the NMR time scale wi th a value of <200 s(-1) at 5 degrees C for the corresponding first-order r ate constants. In wild-type Ran, the equilibrium constant K' between the tw o states is 0.7 at 278 K, is different for various mutants, and is strongly dependent on the temperature. The standard enthalpy Delta H degrees and th e standard entropy Delta S degrees for the conformational transitions deter mined from the NMR spectra are as follows: Delta H degrees = 37 kJ mol(-1) and Delta S degrees = 130 J mol(-1) K-1 for wild-type Ran.GTP. In complex w ith the Ran-binding protein RanBP1, one of the Ran.GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotide excha nge factor protein RCC1 was also studied by P-31 NMR spectroscopy. In the p resence of nucleotide, the ternary complex of Ran.nucleotide.RCC1, an inter mediate in the guanine nucleotide exchange reaction, could be observed. A m odel for the conformational transition of Ran.GTP is proposed where the two states observed are caused by the structural flexibility of the effector l oop of Ran; in solution, state 2 resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.