Conformational States of the Nuclear GTP-Binding Protein Ran and Its Complexes with the Exchange Factor RCC1 and the Effector Protein RanBP1
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- 作者：Matthias Geyer ; Ralf Assheuer ; Christian Klebe ; Jü ; rgen Kuhlmann ; Jö ; rg Becker ; Alfred Wittinghofer ; and Hans Robert Kalbitzer
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：August 31, 1999
- 年：1999
- 卷：38
- 期：35
- 页码：11250 - 11260
- 全文大小：167K
- 年卷期：v.38,no.35(August 31, 1999)
- ISSN：1520-4995
文摘
It has been shown before by 31P NMR that Ras bound to the nonhydrolyzable GTP analogueguanosine 5'-O-(
,
-imidotriphosphate) (GppNHp) exists in two conformations which are rapidlyinterconverting with a rate constant of 3200 s-1 at 30 
C [Geyer, M., et al. (1996) Biochemistry 35,10308-10320]. Here we show that Ran complexed with GTP also exists in two conformational states, 1and 2, which can be directly inferred from the occurrence of two 31P NMR resonance lines for the-phosphate group of bound GTP. The exchange between the two states is slow on the NMR time scalewith a value of <200 s-1 at 5 C for the corresponding first-order rate constants. In wild-type Ran, theequilibrium constant K' between the two states is 0.7 at 278 K, is different for various mutants, and isstrongly dependent on the temperature. The standard enthalpy 
H and the standard entropy S for theconformational transitions determined from the NMR spectra are as follows: H = 37 kJ mol-1 andS = 130 J mol-1 K-1 for wild-type Ran·GTP. In complex with the Ran-binding protein RanBP1, oneof the Ran·GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotideexchange factor protein RCC1 was also studied by 31P 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 twostates observed are caused by the structural flexibility of the effector loop of Ran; in solution, state 2resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.
,-imidotriphosphate) (GppNHp) exists in two conformations which are rapidlyinterconverting with a rate constant of 3200 s-1 at 30 C [Geyer, M., et al. (1996) Biochemistry 35,10308-10320]. Here we show that Ran complexed with GTP also exists in two conformational states, 1and 2, which can be directly inferred from the occurrence of two 31P NMR resonance lines for the-phosphate group of bound GTP. The exchange between the two states is slow on the NMR time scalewith a value of <200 s-1 at 5 C for the corresponding first-order rate constants. In wild-type Ran, theequilibrium constant K' between the two states is 0.7 at 278 K, is different for various mutants, and isstrongly dependent on the temperature. The standard enthalpy H and the standard entropy S for theconformational transitions determined from the NMR spectra are as follows: H = 37 kJ mol-1 andS = 130 J mol-1 K-1 for wild-type Ran·GTP. In complex with the Ran-binding protein RanBP1, oneof the Ran·GTP conformations (state 2) is stabilized. The interaction of Ran with the guanine nucleotideexchange factor protein RCC1 was also studied by 31P 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 twostates observed are caused by the structural flexibility of the effector loop of Ran; in solution, state 2resembles the GTP-bound form found in the crystal structure of the Ran-RanBP complex.