Many regulatory PPP1R subunits join few catalytic PP1c subunits to mediate phosphoserine and phosphothreonine dephosphorylation in metazoans. Regulatory subunits engage the surface of PP1c, locally affecting flexible access of the phosphopeptide to the active site. However, catalytic efficiency of holophosphatases towards their phosphoprotein substrates remains unexplained. Here we present a cryo-EM structure of the tripartite PP1c–PPP1R15A–G-actin holophosphatase that terminates signaling in the mammalian integrated stress response (ISR) in the pre-dephosphorylation complex with its substrate, translation initiation factor 2α (eIF2α). G-actin, whose essential role in eIF2α dephosphorylation is supported crystallographically, biochemically and genetically, aligns the catalytic and regulatory subunits, creating a composite surface that engages the N-terminal domain of eIF2α to position the distant phosphoserine-51 at the active site. Substrate residues that mediate affinity for the holophosphatase also make critical contacts with eIF2α kinases. Thus, a convergent process of higher-order substrate recognition specifies functionally antagonistic phosphorylation and dephosphorylation in the ISR.

许多调节 PPP1R 亚基加入少数催化 PP1c 亚基以介导后生动物中的磷酸丝氨酸和磷酸苏氨酸去磷酸化。调节亚基与 PP1c 的表面结合，局部影响磷酸肽灵活进入活性位点。然而，全磷酸酶对其磷蛋白底物的催化效率仍未得到解释。在这里，我们展示了三联 PP1c–PPP1R15A–G-肌动蛋白全磷酸酶的冷冻电镜结构，该酶在预去磷酸化复合物及其底物翻译起始因子 2α (eIF2α) 中终止哺乳动物综合应激反应 (ISR) 中的信号传导。G-肌动蛋白，其在 eIF2α 去磷酸化中的重要作用在晶体学、生物化学和遗传学上得到支持，排列催化和调节亚基，创建一个复合表面，该表面与 eIF2α 的 N 末端结构域结合，以将远处的磷酸丝氨酸 51 定位在活性位点。介导全磷酸酶亲和力的底物残基也与 eIF2α 激酶发生关键接触。因此，高阶底物识别的收敛过程指定了 ISR 中的功能性拮抗磷酸化和去磷酸化。