Créchet J B, Bernardi A, Parmeggiani A
Groupe de Biophysique-Equipe 2, Ecole Polytechnique, F-91128 Palaiseau Cedex, France.
J Biol Chem. 1996 Jul 19;271(29):17234-40. doi: 10.1074/jbc.271.29.17234.
The interaction of Saccharomyces cerevisiae Ras2p with the catalytic domain of the GDP/GTP exchange factors (GEFs) mouse CDC25(Mm), yeast Cdc25p, and Sdc25p was analyzed by introducing the substitution R80D/N81D into Ras2p S24N, a mutant that is shown to interfere with the Ras2p wild type (wt)-GEF interaction by forming a stable complex. The triple mutant, like Ras2p R80D/N81D, did not interfere with the action of GEF on Ras2p wt (or H-Ras p21) and was unable to form a stable complex with GEF. The GEF stimulation of the nucleotide dissociation of the triple mutant was virtually abolished and strongly decreased with the double mutant. The affinity of Ras2p S24N/R80D/N81D for GDP and GTP was decreased 3 and 4 orders of magnitude, respectively, like that of Ras2p S24N, whereas the double mutant behaved as Ras2p wt. Like Ras2p S24N and unlike Ras2p R80D/N81D, the GTP-bound triple mutant did not activate adenylyl cyclase. Thus, the triple mutant and Ras2p S24N have opposite properties toward the binding to GEF but similarly modified behaviors toward GDP, GTP, and adenylyl cyclase. This work emphasizes the determinant role of the distal switch II region of Ras2p for the interaction with GEF and the different structural background of the interaction with adenylyl cyclase.
通过将R80D/N81D替换引入Ras2p S24N（一种已显示通过形成稳定复合物干扰Ras2p野生型（wt）-鸟苷酸交换因子（GEF）相互作用的突变体），分析了酿酒酵母Ras2p与GDP/GTP交换因子（GEF）小鼠CDC25（Mm）、酵母Cdc25p和Sdc25p催化结构域的相互作用。该三重突变体与Ras2p R80D/N81D一样，不干扰GEF对Ras2p wt（或H-Ras p21）的作用，并且无法与GEF形成稳定复合物。三重突变体的核苷酸解离的GEF刺激实际上被消除，而双重突变体则显著降低。Ras2p S24N/R80D/N81D对GDP和GTP的亲和力分别降低了3个和4个数量级，与Ras2p S24N一样，而双重突变体的表现与Ras2p wt相同。与Ras2p S24N一样但与Ras2p R80D/N81D不同，结合GTP的三重突变体不激活腺苷酸环化酶。因此，三重突变体和Ras2p S24N在与GEF结合方面具有相反的特性，但在与GDP、GTP和腺苷酸环化酶的相互作用行为上有类似的改变。这项工作强调了Ras2p远端开关II区域在与GEF相互作用中的决定性作用以及与腺苷酸环化酶相互作用的不同结构背景。
