Department of Immunology and Infectious Diseases, Harvard TH Chan School of Public Health, Boston, Massachusetts, USA.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
J Bacteriol. 2021 Jan 25;203(4). doi: 10.1128/JB.00608-20.
The ClpP1P2 proteolytic complex is essential in Proteolysis by ClpP1P2 requires an associated ATPase, either ClpX or ClpC1. Here, we sought to define the unique contributions of the ClpX ATPase to mycobacterial growth. We formally demonstrated that ClpX is essential for mycobacterial growth, and to understand its essential functions, we identified ClpX-His-interacting proteins by pulldown and tandem mass spectrometry. We found an unexpected association between ClpX and proteins involved in DNA replication, and we confirm a physical association between ClpX and the essential DNA maintenance protein single-stranded-DNA binding protein (SSB). Purified SSB is not degraded by ClpXP1P2; instead, SSB enhances ATP hydrolysis by ClpX and degradation of the model substrate GFP-SsrA by ClpXP1P2. This activation of ClpX is mediated by the C-terminal tail of SSB, which had been implicated in the activation of other ATPases associated with DNA replication. Consistent with the predicted interactions, depletion of transcript perturbs DNA replication. These data reveal that ClpX participates in DNA replication and identify the first activator of ClpX in mycobacteria. Tuberculosis, caused by , imposes a major global health burden, surpassing HIV and malaria in annual deaths. The ClpP1P2 proteolytic complex and its cofactor ClpX are attractive drug targets, but their precise cellular functions are unclear. This work confirms ClpX's essentiality and describes a novel interaction between ClpX and SSB, a component of the DNA replication machinery. Further, we demonstrate that a loss of ClpX is sufficient to interrupt DNA replication, suggesting that the ClpX-SSB complex may play a role in DNA replication in mycobacteria.
ClpP1P2 蛋白水解复合物在蛋白水解中必不可少，需要一种相关的 ATP 酶，即 ClpX 或 ClpC1。在这里，我们试图确定 ClpX ATP 酶对分枝杆菌生长的独特贡献。我们正式证明 ClpX 对分枝杆菌的生长是必不可少的，为了了解其基本功能，我们通过下拉和串联质谱鉴定了 ClpX-His 相互作用蛋白。我们发现 ClpX 与参与 DNA 复制的蛋白质之间存在出乎意料的关联，并且我们确认了 ClpX 与必需的 DNA 维持蛋白单链结合蛋白 (SSB) 之间的物理关联。纯化的 SSB 不会被 ClpXP1P2 降解；相反，SSB 增强了 ClpX 的 ATP 水解，并增强了 ClpXP1P2 对模型底物 GFP-SsrA 的降解。这种 ClpX 的激活是由 SSB 的 C 端尾巴介导的，该尾巴已被牵连到与 DNA 复制相关的其他 ATP 酶的激活中。与预测的相互作用一致， 转录物的耗尽会扰乱 DNA 复制。这些数据表明 ClpX 参与 DNA 复制，并确定了分枝杆菌中 ClpX 的第一个激活剂。由 引起的结核病对全球健康造成了重大负担，在年度死亡人数上超过了 HIV 和疟疾。ClpP1P2 蛋白水解复合物及其辅助因子 ClpX 是有吸引力的药物靶点，但它们的确切细胞功能尚不清楚。这项工作证实了 ClpX 的必要性，并描述了 ClpX 与 SSB 之间的新相互作用，SSB 是 DNA 复制机制的一部分。此外，我们证明 ClpX 的缺失足以中断 DNA 复制，这表明 ClpX-SSB 复合物可能在分枝杆菌的 DNA 复制中发挥作用。
