结核分枝杆菌 Ku C 端通过结构域特异性互作调控 LigD 活性
The Mycobacterium tuberculosis Ku C-terminus orchestrates LigD activity through domain-specific interactions
结核分枝杆菌 Ku C 端与 LigD 的聚合酶结构域和连接酶结构域形成二分界面，既刺激连接酶活性又抑制模板依赖的聚合酶活性。研究结合 NMR 波谱、结构建模和突变分析，锁定 Ku 的 E246、V248、S258、K260、N266 以及 LigD 聚合酶域 D162、V194、R198 和连接酶域 D522、K579、L580 为介导互作的关键残基。功能实验显示，Ku 通过同时接触两个结构域刺激 LigD 连接酶活性，却减弱其模板依赖的聚合酶活性，这与铜绿假单胞菌同源蛋白的既往结果相反。
来源：Nucleic Acids Research · doi.org
Bacterial non-homologous end joining (NHEJ) is a key DNA double-strand break (DSB) repair pathway that relies on the Ku–LigD complex to alleviate genomic instability. The Mycobacterium tuberculosis Ku C-terminus has been highlighted for its role in LigD recruitment to DSBs and stimulation of LigD ligase activity. However, it remains unclear how the Ku C-terminus interacts with and potentially influences other LigD activities. Here, we combine NMR spectroscopy, structural modelling, and mutational analysis to define the interaction interface between the Ku C-terminus and LigD. We identify critical residues in Ku (E246, V248, S258, K260, and N266) and the LigD polymerase (D162, V194, R198) and ligase (D522, K579, L580) domains that mediate this interaction. Functional assays reveal that Ku stimulates LigD ligase activity through contacts with both polymerase and ligase domains, while Ku attenuates template-dependent polymerase activity, contrasting previous studies in Pseudomonas aeruginosa homologs. Disrupting the Ku–LigD interface, either through Ku or LigD mutations, abolishes ligase stimulation and restores polymerase activity, highlighting a dual regulatory mechanism. Our data supports a model where Ku’s C-terminal region forms a bipartite interface with LigD to balance repair activity. These findings provide mechanistic insight into the Ku–LigD repair mechanism and uncovers species-specific differences in bacterial NHEJ.