Conformational Transitions in p21ras and in Its Complexes with the Effector Protein Raf-RBD and the GTPase Activating Protein GAP
详细信息 查看全文
- 作者：Matthias Geyer ; Thomas Schweins ; Christian Herrmann ; Thomas Prisner ; Alfred Wittinghofer ; and Hans Robert Kalbitzer
- 刊名：Biochemistry
- 出版年：1996
- 出版时间：August 13, 1996
- 年：1996
- 卷：35
- 期：32
- 页码：10308 - 10320
- 全文大小：522K
- 年卷期：v.35,no.32(August 13, 1996)
- ISSN：1520-4995
文摘
31P NMR revealed that the complex ofp21ras with the GTP analogGppNHp·Mg2+ exists intwo conformational states, states 1 and 2. In wild-typep21ras the equilibrium constant
betweenthetwo states is 1.09. The population of these states is differentfor various mutants but independent oftemperature. The activation enthalpy
H
and activation entropyS for the conformationaltransitionswere determined by full-exchange matrix analysis for wild-typep21ras andp21ras(S65P). For thewild-type protein one obtains H = 89 ± 2 kJmol-1 and S =102 ± 20 J mol-1K-1 and for the mutantprotein H = 93 ± 7 kJmol-1 and S =138 ± 30 J mol-1K-1. The study of variousp21ras mutantssuggests that the two states correspond to different conformations ofloop L2, with Tyr-32 in two differentpositions relative to the bound nucleotide. High-field EPR at 95GHz suggests that the observedconformational transition does not directly influence the coordinationsphere of the protein-bound metalion. The influence of this transition on loop L4 was studied by1H NMR with mutants E62H and E63H.There was no indication that L4 takes part in the transitiondescribed in L2, although a reversibleconformational change could be induced by decreasing the pH value.The exchange between the twostates is slow on the NMR time scale (<10s-1); at approximately pH 5 the population ofthe two statesis equal. The interaction ofp21ras-triphosphate complexes with theRas-binding domain (RBD) of theeffector protein c-Raf-1, Raf-RBD, and with the GTPase activatingprotein GAP was studied by 31P NMRspectroscopy. In complex with Raf-RBD the second conformation ofp21ras (state 2) is stabilized.Inthis conformation Tyr-32 is located in close proximity to the phosphategroups of the nucleotide, and the
-phosphate resonance is shifted upfield by 0.7 ppm. Spectraobtained in the presence of GAP suggestthat in the ground state GAP does not interact directly with thenucleotide bound to p21ras and doesnotinduce larger conformational changes in the neighborhood of thenucleotide. The experimental data areconsistent with a picture where GAP accelerates the exchange processbetween the two states andsimultaneously increases the population of state 1 at highertemperature.
betweenthetwo states is 1.09. The population of these states is differentfor various mutants but independent oftemperature. The activation enthalpyH and activation entropyS for the conformationaltransitionswere determined by full-exchange matrix analysis for wild-typep21ras andp21ras(S65P). For thewild-type protein one obtains H = 89 ± 2 kJmol-1 and S =102 ± 20 J mol-1K-1 and for the mutantprotein H = 93 ± 7 kJmol-1 and S =138 ± 30 J mol-1K-1. The study of variousp21ras mutantssuggests that the two states correspond to different conformations ofloop L2, with Tyr-32 in two differentpositions relative to the bound nucleotide. High-field EPR at 95GHz suggests that the observedconformational transition does not directly influence the coordinationsphere of the protein-bound metalion. The influence of this transition on loop L4 was studied by1H NMR with mutants E62H and E63H.There was no indication that L4 takes part in the transitiondescribed in L2, although a reversibleconformational change could be induced by decreasing the pH value.The exchange between the twostates is slow on the NMR time scale (<10s-1); at approximately pH 5 the population ofthe two statesis equal. The interaction ofp21ras-triphosphate complexes with theRas-binding domain (RBD) of theeffector protein c-Raf-1, Raf-RBD, and with the GTPase activatingprotein GAP was studied by 31P NMRspectroscopy. In complex with Raf-RBD the second conformation ofp21ras (state 2) is stabilized.Inthis conformation Tyr-32 is located in close proximity to the phosphategroups of the nucleotide, and the-phosphate resonance is shifted upfield by 0.7 ppm. Spectraobtained in the presence of GAP suggestthat in the ground state GAP does not interact directly with thenucleotide bound to p21ras and doesnotinduce larger conformational changes in the neighborhood of thenucleotide. The experimental data areconsistent with a picture where GAP accelerates the exchange processbetween the two states andsimultaneously increases the population of state 1 at highertemperature.