Geyer M, Schweins T, Herrmann C, Prisner T, Wittinghofer A, Kalbitzer H R
Max-Planck-Institut für medizinische Forschung, Berlin, Germany.
Biochemistry. 1996 Aug 13;35(32):10308-20. doi: 10.1021/bi952858k.
31P NMR revealed that the complex of p21ras with the GTP analog GppNHp.Mg2+ exists in two conformational states, states 1 and 2. In wild-type p21ras the equilibrium constant K1(12) between the two states is 1.09. The population of these states is different for various mutants but independent of temperature. The activation enthalpy delta H ++ and activation entropy delta S ++ for the conformational transitions were determined by full-exchange matrix analysis for wild-type p21ras and p21ras(S65P). For the wild-type protein one obtains delta H ++ = 89 +/- 2 kJ mol-1 and delta S ++ = 102 +/- 20 J mol-1 K-1 and for the mutant protein delta H ++ = 93 +/- 7 kJ mol-1 and delta S ++ = 138 +/- 30 J mol-1 K-1. The study of various p21ras mutants suggests that the two states correspond to different conformations of loop L2, with Tyr-32 in two different positions relative to the bound nucleotide. High-field EPR at 95 GHz suggest that the observed conformational transition does not directly influence the coordination sphere of the protein-bound metal ion. The influence of this transition on loop L4 was studied by 1H NMR with mutants E62H and E63H. There was no indication that L4 takes part in the transition described in L2, although a reversible conformational change could be induced by decreasing the pH value. The exchange between the two states is slow on the NMR time scale (< 10 s-1): at approximately pH 5 the population of the two states is equal. The interaction of p21ras-triphosphate complexes with the Ras-binding domain (RBD) of the effector protein c-Raf-1, Raf-RBD, and with the GTPase activating protein GAP was studied by 31P NMR spectroscopy. In complex with Raf-RBD the second conformation of p21ras (state 2) is stabilized. In this conformation Tyr-32 is located in close proximity to the phosphate groups of the nucleotide, and the beta-phosphate resonance is shifted upfield by 0.7 ppm. Spectra obtained in the presence of GAP suggest that in the ground state GAP does not interact directly with the nucleotide bound to p21ras and does not induce larger conformational changes in the neighborhood of the nucleotide. The experimental data are consistent with a picture where GAP accelerates the exchange process between the two states and simultaneously increases the population of state 1 at higher temperature.
31P核磁共振显示，p21ras与GTP类似物GppNHp·Mg2+的复合物存在两种构象状态，即状态1和状态2。在野生型p21ras中，这两种状态之间的平衡常数K1(12)为1.09。不同突变体中这些状态的比例不同，但与温度无关。通过对野生型p21ras和p21ras(S65P)进行全交换矩阵分析，确定了构象转变的活化焓ΔH++和活化熵ΔS++。对于野生型蛋白，得到ΔH++ = 89±2 kJ/mol和ΔS++ = 102±20 J/(mol·K)，对于突变蛋白，ΔH++ = 93±7 kJ/mol和ΔS++ = 138±30 J/(mol·K)。对各种p21ras突变体的研究表明，这两种状态对应于环L2的不同构象，Tyr-32相对于结合的核苷酸处于两个不同位置。95 GHz的高场电子顺磁共振表明，观察到的构象转变不会直接影响与蛋白结合的金属离子的配位球。通过对突变体E62H和E63H进行1H核磁共振研究了这种转变对环L4的影响。没有迹象表明L4参与了L2中描述的转变，尽管降低pH值可诱导可逆的构象变化。在核磁共振时间尺度上（<10 s-1），两种状态之间的交换很慢：在大约pH 5时，两种状态的比例相等。通过31P核磁共振光谱研究了p21ras-三磷酸复合物与效应蛋白c-Raf-1的Ras结合结构域（RBD）、Raf-RBD以及与GTP酶激活蛋白GAP的相互作用。与Raf-RBD形成复合物时，p21ras的第二种构象（状态2）得以稳定。在这种构象中，Tyr-32位于靠近核苷酸磷酸基团的位置，β-磷酸共振向上位移0.7 ppm。在GAP存在下获得的光谱表明，在基态下，GAP不会直接与结合到p21ras的核苷酸相互作用，也不会在核苷酸附近诱导较大的构象变化。实验数据与这样一种情况一致，即GAP加速了两种状态之间的交换过程，同时在较高温度下增加了状态1的比例。
