Azuma Y, Renault L, García-Ranea J A, Valencia A, Nishimoto T, Wittinghofer A
Department of Molecular Biology, Kyushu University, Fukuoka, Japan.
J Mol Biol. 1999 Jun 18;289(4):1119-30. doi: 10.1006/jmbi.1999.2820.
RCC1, the regulator of chromosome condensation, is the guanine nucleotide exchange factor (GEF) for the nuclear Ras-like GTP-binding protein Ran. Its structure was solved by X-ray crystallography and revealed a seven-bladed beta-propeller, one side of which was proposed to be the interaction site with Ran. To gain more insight into this interaction, alanine mutagenesis studies were performed on conserved residues on the surface of the structure. Purified mutant proteins were analysed by steady-state kinetic analysis of their GEF activities towards Ran. A number of residues were identified whose mutation affected either the KMor kcatof the overall reaction, or had no effect. Mutants were further analysed by plasmon surface resonance in order to get more information on individual steps of the complex reaction pathway. Ran-GDP was coupled to the sensor chip and reacted with RCC1 mutants to categorise them into different groups, demonstrating the usefulness of plasmon surface resonance in the study of complex multi-step kinetic processes. A docking solution of Ran-RCC1 structures in combination with sequence analysis allows prediction of the site of interaction between RCC1 and Ran and proposes a model for the Ran-RCC1 structure which corresponds to and extends the biochemical data. Three invariant residues which most severely affect the kcatof the reaction, D128, D182 and H304, are located in the centre of the Ran-RCC1 interface and interfere with switch II and the phosphate binding area. The structural model suggests that different guanine nucleotide exchange factors use a similar interaction site on their respective GTP-binding proteins, but that the molecular mechanisms for the release of nucleotides are likely to be different.
RCC1是染色体凝聚调节因子，是核内类Ras鸟苷三磷酸（GTP）结合蛋白Ran的鸟嘌呤核苷酸交换因子（GEF）。其结构通过X射线晶体学解析得出，为一个七叶β-螺旋桨结构，其一侧被认为是与Ran的相互作用位点。为了更深入了解这种相互作用，对该结构表面的保守残基进行了丙氨酸诱变研究。通过对其针对Ran的GEF活性进行稳态动力学分析，对纯化的突变蛋白进行了分析。鉴定出了一些残基，其突变要么影响了整体反应的米氏常数（Km）或催化常数（kcat），要么没有影响。为了获取关于复杂反应途径各个步骤的更多信息，通过表面等离子体共振对突变体进行了进一步分析。将Ran-GDP偶联到传感芯片上，并与RCC1突变体反应，以便将它们分类为不同的组，这证明了表面等离子体共振在研究复杂多步动力学过程中的有用性。Ran-RCC1结构的对接解决方案与序列分析相结合，可以预测RCC1与Ran之间的相互作用位点，并提出一个与生化数据相符且有所扩展的Ran-RCC1结构模型。三个对反应的kcat影响最为严重的不变残基，即D128、D182和H304，位于Ran-RCC1界面的中心，并干扰开关II和磷酸盐结合区域。该结构模型表明，不同的鸟嘌呤核苷酸交换因子在其各自的GTP结合蛋白上使用相似的相互作用位点，但核苷酸释放的分子机制可能不同。
