Protein phosphorylation is a pivotal post-translational modification modulating various cellular processes. In Gram-positive bacteria, the protein arginine kinase McsB, along with its activator McsA, has a key role in labeling misfolded and damaged proteins during stress. However, the activation mechanism of McsB by McsA remains elusive. Here we report the cryo-electron microscopy structure of a tetrameric McsA–McsB complex at 3.41 Å resolution. Biochemical analysis indicates that the homotetrameric assembly is essential for McsB’s kinase activity. The conserved C-terminal zinc finger of McsA interacts with an extended loop in McsB, optimally orienting a critical catalytic cysteine residue. In addition, McsA binding decreases the CtsR’s affinity for McsB, enhancing McsB’s kinase activity and accelerating the turnover rate of CtsR phosphorylation. Furthermore, McsA binding also increases McsB’s thermostability, ensuring its activity under heat stress. These findings elucidate the structural basis and activation mechanism of McsB in stress response.

蛋白质磷酸化是调节各种细胞过程的关键翻译后修饰。在革兰氏阳性细菌中，蛋白质精氨酸激酶 McsB 及其激活剂 McsA 在标记应激期间错误折叠和受损的蛋白质方面发挥着关键作用。然而，McsA 激活 McsB 的机制仍不清楚。在这里，我们报告了四聚体 McsA-McsB 复合物的冷冻电子显微镜结构，分辨率为 3.41 Å。生化分析表明同源四聚体组装对于 McsB 的激酶活性至关重要。 McsA 的保守 C 端锌指与 McsB 中的延伸环相互作用，从而最佳地定向关键的催化半胱氨酸残基。此外，McsA 结合降低了 CtsR 对 McsB 的亲和力，增强了 McsB 的激酶活性并加速了 CtsR 磷酸化的周转率。此外，McsA 结合还提高了 McsB 的热稳定性，确保其在热应激下的活性。这些发现阐明了McsB在应激反应中的结构基础和激活机制。