CONSORTIUM FOR LARGE MACROMOLECULAR STRUCTURES

大分子结构联盟

• 批准号: 6220516
• 负责人: PAUL B SIGLER
• 金额: $ 1.42万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-15 至 2000-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6220516
• 关键词: bioimaging /biomedical imaging; biological products; biomedical resource; proteins; radiography; structural biology

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.

我们已经解决了小GTP结合的晶体结构 蛋白质 Cdc42 与其 C 端结构域形成复合物 GTP酶激活蛋白（GAP）通过MAD pahsing和a的组合 分子替代溶液。 最终模型现已细化为2.1 Rfree 的分辨率为 25.9%，显示 AlF3 的存在 分子在活性位点。 复合体的结构表明 GAP 也本质上与 Cdc42 中的开关 I 和 II 环结合 作为螺旋H3的末端。 通过这样做，GAP 稳定了一个 Gln61 的特殊构象，一种已被发现的残基 对所有 G 蛋白都是必需的。 与此同时，GAP 引入了 将保守的精氨酸残基 (R305) 插入活性位点以稳定 GTP 上的负电荷形成。 侧链酰胺基团 R305 的一个与一个氟原子紧密接触并与 β-γ-磷酸盐-桥接氧。 这一观察结果表明 负电荷集中在γ-磷酸盐和离开 组（GDP）。 因此，过渡状态具有混合状态 结合和解离结构。 进一步研究作用 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，AlF3 分子也被发现于 活性位点。 G蛋白和GAP之间的界面 没有改变。 来自 Cdc42 的 Tyr32，它在野外稳定 R305 型突变体，已改变其构象，现在紧密结合 AlF3。 突变体复合物中的 Gln61 被认为更加灵活 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， 功能是稳定 G 蛋白的开关结构域并 引入精氨酸残基以稳定过渡形成。