State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China.
State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China; Joint International Research Laboratory of Metabolic & Developmental Sciences, Shanghai Jiao Tong University, Shanghai 200240, China.
Int J Biol Macromol. 2024 Oct;277(Pt 1):134145. doi: 10.1016/j.ijbiomac.2024.134145. Epub 2024 Jul 24.
Bacterial defense-associated sirtuin 2 (DSR2) proteins harbor an N-terminal sirtuin (SIR2) domain degrading NAD. DSR2 from Bacillus subtilis 29R is autoinhibited and unable to hydrolyze NAD in the absence of phage infection. A tail tube protein (TTP) of phage SPR activates the DSR2 while a DSR2-inhibiting protein of phage SPbeta, known as DSAD1 (DSR anti-defense 1), inactivates the DSR2. Although DSR2 structures in complexed with TTP and DSAD1, respectively, have been reported recently, the autoinhibition and activation mechanisms remain incompletely understood. Here, we present cryo-electron microscopy structures of the DSR2-NAD complex in autoinhibited state and the in vitro assembled DSR2-TFD (TTP tube-forming domain) complex in activated state. The DSR2-NAD complex reveals that the autoinhibited DSR2 assembles into an inactive tetramer, binding NAD through a distinct pocket situated outside active site. Binding of TFD into cavities within the sensor domains of DSR2 triggers a conformational change in SIR2 regions, activating its NADase activity, whereas the TTP β-sandwich domain (BSD) is flexible and does not contribute to the activation process. The activated form of DSR2 exists as tetramers and dimers, with the tetramers exhibiting more NADase activity. Overall, our results extend the current understanding of autoinhibition and activation of DSR2 immune proteins.
细菌防御相关的沉默调节蛋白 2（DSR2）蛋白含有一个 N 端的沉默调节蛋白（SIR2）结构域，可降解 NAD。枯草芽孢杆菌 29R 的 DSR2 自身受到抑制，在没有噬菌体感染的情况下无法水解 NAD。噬菌体 SPR 的尾管蛋白（TTP）激活 DSR2，而噬菌体 SPbeta 的 DSR2 抑制蛋白，称为 DSAD1（DSR 抗防御 1），则使 DSR2 失活。尽管最近分别报道了 DSR2 与 TTP 和 DSAD1 复合物的结构，但自动抑制和激活机制仍不完全清楚。在这里，我们展示了处于自动抑制状态的 DSR2-NAD 复合物和体外组装的 DSR2-TFD（TTP 管形成结构域）复合物的冷冻电镜结构。DSR2-NAD 复合物表明，自动抑制的 DSR2 组装成一个无活性的四聚体，通过位于活性位点之外的独特口袋结合 NAD。TFD 结合到 DSR2 的传感器结构域内的腔中，触发 SIR2 区域的构象变化，激活其 NADase 活性，而 TTP β-夹心结构域（BSD）是灵活的，不参与激活过程。激活形式的 DSR2 以四聚体和二聚体存在，其中四聚体具有更高的 NADase 活性。总的来说，我们的结果扩展了对 DSR2 免疫蛋白自动抑制和激活的现有认识。
