Conformational States of the Nuclear GTP-Binding Protein Ran and Its Complexes with the Exchange Factor RCC1 and the Effector Protein RanBP1

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.

Conformational States of the Small G Protein Arf-1 in Complex with the Guanine Nucleotide Exchange Factor ARNO-Sec7

“…In Ras as well as in Ran, the complexes with nucleoside triphosphates occur in two conformational states as has been shown by 31 P NMR spectroscopy (15,16). In Ran, the two states are easy to observe with GTP, where two sets of signals for the bound GTP are observed (16).…”

“…In Ran, the two states are easy to observe with GTP, where two sets of signals for the bound GTP are observed (16). Especially the ␥-phosphate signals of the two states are well separated by 1.68 ppm.…”

“…For the small G proteins Ran (16), Ras (16,17), and EF-Tu (14, 18), a strong downfield shift of the ␤-phosphate resonance and a moderate downfield shift of the ␣-phosphate resonance of GDP after binding to the protein is typical. Besides shifts caused by the anisotropy of the magnetic susceptibility of nearby residues (e.g.…”

“…From the x-ray structure, possible candidates can be derived; the main candidate is Lys 30 in Arf1, which is conserved in small GTPases. Lys 30 corresponds to Lys 16 in Ras, Lys 23 in Ran, and Lys 24 in EF-Tu.…”

“…brefeldin A (11), membrane lipids (9)) but have not been trapped by crystallography. Phosphorus NMR was shown to be an alternative to characterize different steps of GTP-induced conformations in effector recognition (15)(16)(17) as well as of GDP dissociation (16) of small G proteins. At a millimolar concentration of protein and nucleotide used, one can detect weak ternary complexes between a small G protein, nucleotide, and exchange factor.…”