Division of Molecular Medicine, Beckman Research Institute of the City of Hope, Duarte, CA 91010, USA.
J Biol Chem. 2010 Jul 30;285(31):23732-8. doi: 10.1074/jbc.M110.114660. Epub 2010 May 25.
Post-translational modifications by ubiquitin-like proteins are among the most important mechanisms for regulating a wide variety of cellular functions. In these modifications an E1 enzyme activates each ubiquitin-like protein (Ubl) by adenylation of the Ubl C-terminal COOH group and then forms a thioester bond with the adenylated C-terminal COOH group of the Ubl. Previous x-ray crystallography studies revealed a conserved zinc motif in the SUMO and NEDD8 E1; however, the function of this Zn(2+) motif is unclear. In this study, using quantitative ATP:PPi isotope exchange assays in combination with site-directed mutagenesis, we show that the conserved Zn(2+) motif in the SUMO E1 is important for SUMO adenylation and is critical for the E1 pseudo-ordered substrate binding mechanism. Furthermore, Zn(2+) motif mutants showed significantly reduced k(cat) values for ATP:PPi isotope exchange assays, suggesting that the Zn(2+) motif is important in binding and preventing SUMO adenylate from dissociating from E1 before formation of the thioester conjugate. Because the Zn(2+) motif is located in a cross-over loop that is known to have conformational flexibility, the results described here suggest that this cross-over loop interacts with Ubl in the multistep, dynamic process of Ubl activation by E1s.
泛素样蛋白的翻译后修饰是调节多种细胞功能的最重要机制之一。在这些修饰中，E1 酶通过泛素样蛋白 (Ubl) C 末端 COOH 基团的腺苷酸化激活每个泛素样蛋白 (Ubl)，然后与 Ubl 的腺苷酸化 C 末端 COOH 基团形成硫酯键。先前的 X 射线晶体学研究揭示了 SUMO 和 NEDD8 E1 中的保守锌模体；然而，该 Zn(2+)模体的功能尚不清楚。在这项研究中，我们使用定量 ATP:PPi 同位素交换测定法结合定点突变，表明 SUMO E1 中的保守 Zn(2+)模体对 SUMO 腺苷酸化很重要，并且对 E1 拟有序底物结合机制至关重要。此外，Zn(2+)模体突变体的 ATP:PPi 同位素交换测定的 k(cat) 值显着降低，表明 Zn(2+)模体在结合和防止 SUMO 腺苷酸从 E1 上解离形成硫酯键之前很重要。由于 Zn(2+)模体位于交叉环中，已知该环具有构象灵活性，因此这里描述的结果表明，该交叉环在 E1 激活 Ubl 的多步动态过程中与 Ubl 相互作用。
