CONSORTIUM FOR LARGE MACROMOLECULAR STRUCTURES

我们已经解决了小分子GTP结合的晶体结构 蛋白质CDC42与其C-末端结构域的复合体 GTP酶激活蛋白(GAP)由MAD PACHING和A 分子置换溶液。最终的模型现在改进为2.1 RFree的分辨率为25.9%，并显示存在AlF3 活性部位的分子。建筑群的结构显示了 Gap实质上也绑定到CDC42中的Switch I和II环路 作为螺旋H3的末端。在这样做的过程中，差距稳定了一个 Gln61的特殊构象，已发现的残基是 对所有G蛋白来说都是必不可少的。与此同时，这一差距带来了一个 将精氨酸残基(R305)保守到活性部位以稳定 GTP上负电荷的形成。侧链酰胺基团 的R305与一个氟原子和与 β-伽马-磷酸-桥联-氧气。这一观察表明， 负电荷局限在伽马-磷酸盐上和离开时 集团(国内生产总值)。因此，过渡状态具有混合的 联想结构和解离结构。为进一步研究其作用， R305，我们解决了相同复合体的结构，但使用了 GAP(R305A)突变体。令我们惊讶的是，还发现了一个AlF3分子 活动站点。G蛋白和GAP之间的界面有 没有改变。来自CDC42的Tyr32，它在野外稳定R305 类型突变，已经改变了它的构象，现在与 AlF3。突变复合体中的Gln61被认为更灵活 与亲核攻击水的相互作用较弱。 综上所述，这些结果表明GAP是一个双分子， 其功能是稳定G蛋白的开关域，并 引入精氨酸残基以稳定过渡形成。

We have solved the crystal structure of the small GTP-binding protein Cdc42 in complex with a C-terminal domain of its GTPase-activating protein (GAP) by a combination of MAD pahsing and a molecular replacement solution. The final model is now refined to 2.1 A resolution to an Rfree of 25.9% and shows the presence of an AlF3 molecule in the active site. The structure of the complex shows the GAP to bind essentially to the switch I and II loops in Cdc42 as well as the end of helix H3. In doing so, the GAP stabilizes one particular conformation of Gln61, a residue that has been found to be essential to all G-proteins. At the same time, the GAP introduces a conserved arginine residue (R305) into the active site to stabilize the negative charge formation on the GTP. The side chain amide groups of R305 are in close contact with one fluorine atom and with the beta-gamma-phosphate-bridging-oxygen. This observation shows that the negative charge is localized on the gamma-phosphate and on the leaving group (the GDP). As a consequence, the transition state has a mixed associative and dissociative structure. To further study the role of R305, we solved the structure of the same complex but with a GAP(R305A) mutant. To our surprise an AlF3 molecule was also found in the active site. The interface between the G-protein and the GAP has not changed. Tyr32 from Cdc42, which was stabilizing R305 in the wild type mutant, has changed its conformation and is now tightly bound to the AlF3. Gln61 in the mutant complex, is seen to be more flexible and to have weaker interactions with the nucleophilic attacking water. Taken together, these results show that GAP is a dual molecule which function is to stabilize the switch domains of the G-protein and to introduce an arginine residue to stabilize the transition formation.