CONSORTIUM FOR LARGE MACROMOLECULAR STRUCTURES

我们已经解决了小GTP结合的晶体结构， 蛋白Cdc 42与其C-末端结构域复合， GTPase-activating protein（GAP），通过MAD pahsing和 分子置换液 最终的模型现在被细化到2.1 Rfree的分辨率为25.9%，显示存在AlF 3 分子在活性部位。 复杂的结构表明， GAP也基本上结合Cdc 42中的开关I和II环 作为螺旋H3的末端。 在这样做的时候差距稳定了一个 Gln 61的特殊构象，已发现Gln 61是 对所有G蛋白都至关重要。 与此同时，差距引入了一个 保守的精氨酸残基（R305）进入活性位点，以稳定 GTP上的负电荷形成。 侧链酰胺基团 的R305与一个氟原子紧密接触，并且与 β-γ-磷酸桥氧。 这一观察表明， 负电荷位于γ-磷酸盐上， GDP（GDP）。 因此，过渡状态具有混合的 联想结构和分离结构。 为了进一步研究 R305，我们解决了相同的复杂的结构，但与 GAP（R305 A）突变体。 令我们惊讶的是，在一个实验室中也发现了AlF 3分子。 活动现场。 G蛋白和差距之间的界面 更改的情况下也 来自Cdc 42的Tyr 32，其在野生环境中稳定R305 型突变体，已经改变了它的构象，现在是紧密结合， AlF3。 Gln 61在突变复合体中，被认为是更灵活的 并且与亲核攻击水具有较弱的相互作用。 总之，这些结果表明GAP是一种双分子， 功能是稳定G蛋白的开关结构域， 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.