CONSORTIUM FOR LARGE MACROMOLECULAR STRUCTURES

大分子结构联盟

• 批准号: 6667821
• 负责人: PAUL B SIGLER
• 金额: $ 14.27万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-30 至 2003-08-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6667821
• 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.

我们解决了小GTP结合的晶体结构 蛋白Cdc42与其C末端结构域复合 GTPase激活蛋白（GAP）通过疯狂的Pahsing和A的组合 分子替代溶液。 最终模型现在已完善为2.1 RFRE的分辨率为25.9％，并显示了ALF3的存在 活性位点中的分子。 复合物的结构显示 差距也基本上与CDC42中的开关I和II循环结合 作为Helix H3的末端。 这样，差距稳定了一个 GLN61的特殊构象，已被发现的残基 对于所有G蛋白至关重要。 同时，差距引入了 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。 侧链酰胺组 R305与一个氟原子密切接触，并与 β-γ-磷酸 - 桥氧。 这个观察结果表明 负电荷位于γ-磷酸盐和离开 组（GDP）。 结果，过渡状态混合 联想和分离结构。 进一步研究 R305，我们解决了同一复合物的结构，但 间隙（R305a）突变体。 令我们惊讶的是，在 活跃的站点。 G蛋白和差距之间的接口具有 没有改变。 cdc42的tyr32，它在野外稳定R305 类型突变体，已改变其构象，现在已紧密绑定到 alf3。 突变体复合物中的GLN61被认为更灵活 并与亲核攻击水的相互作用较弱。 综上所述，这些结果表明差距是双分子 功能是稳定G蛋白的开关域，然后 引入精氨酸残基来稳定过渡形成。