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  • Title: Conformational states of the nuclear GTP-binding protein Ran and its complexes with the exchange factor RCC1 and the effector protein RanBP1.
    Author: Geyer M, Assheuer R, Klebe C, Kuhlmann J, Becker J, Wittinghofer A, Kalbitzer HR.
    Journal: Biochemistry; 1999 Aug 31; 38(35):11250-60. PubMed ID: 10471274.
    Abstract:
    It has been shown before by (31)P 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, 10308-10320]. Here we show that Ran complexed with GTP also exists in two conformational states, 1 and 2, which can be directly inferred from the occurrence of two (31)P NMR resonance lines for the gamma-phosphate group of bound GTP. The exchange between the two states is slow on the NMR time scale with a value of <200 s-1 at 5 degrees C for the corresponding first-order rate constants. In wild-type Ran, the equilibrium constant K' between the two states is 0.7 at 278 K, is different for various mutants, and is strongly dependent on the temperature. The standard enthalpy DeltaH degrees and the standard entropy DeltaS degrees for the conformational transitions determined from the NMR spectra are as follows: DeltaH degrees = 37 kJ mol-1 and DeltaS degrees = 130 J mol-1 K-1 for wild-type Ran.GTP. In complex with the Ran-binding protein RanBP1, one of the Ran.GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotide exchange factor protein RCC1 was also studied by (31)P NMR spectroscopy. In the presence of nucleotide, the ternary complex of Ran.nucleotide.RCC1, an intermediate in the guanine nucleotide exchange reaction, could be observed. A model for the conformational transition of Ran.GTP is proposed where the two states observed are caused by the structural flexibility of the effector loop of Ran; in solution, state 2 resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.
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