Fu Wencheng, Wang Yuli, Ge Yashi, Gao Haiyan, Sun Xuan, Deng Zixin, Wang Lianrong, Chen Shi, He Xinyi, Wu Geng
State Key Laboratory of Microbial Metabolism, School of Life Sciences & Biotechnology, the Joint International Research Laboratory of Metabolic & Developmental Sciences MOE, Shanghai Jiao Tong University, Shanghai 200240, China.
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, School of Pharmaceutical Sciences, Wuhan University, Wuhan 430071, China.
Int J Mol Sci. 2025 Jun 16;26(12):5765. doi: 10.3390/ijms26125765.
In a vast variety of prokaryotes such as and , the DNA degradation (Dnd) CDE protein complex (consisting of DndC, DndD, and DndE), together with the DndA/IscS protein and the DndFGH complex, function as a defense barrier to prevent the invasion of non-self-DNA. The DndCDE complex introduces phosphorothioation (PT) modifications into DNA, and the DndFGH complex specifically cleaves non-PT DNA and, thus, restricts horizontal gene transfer and phage invasion. Despite the central importance of the DndCDE complex in DNA PT modification, which catalyzes the oxygen-sulfur swap on DNA, our understanding of this key complex remains poor. Here, we employed protein structure prediction to provide a reasonably reliable prediction of the structure of the DndCDE complex and a 23 bp DNA-DndCDE complex. We found that among the three proteins in the DndCDE complex, DndC, especially its "specificity loop", plays a key role in recognizing the consensus PT modification sequence. In addition, the DndD protein is found to possess a highly conserved structural surface on its globular domain, presumably mediating the dimerization of DndD as well as the DndCDE complex. Furthermore, our normal mode analysis showed that there exists a dynamic transition between a closed and an open state for the DndCDE complex, facilitating its association and release of DNA. Our conclusions were corroborated by biochemical assays using purified proteins. On the whole, we provide molecular insights into the assembly and DNA-recognition mechanism of a central protein complex involved in DNA phosphorothioation.
在各种各样的原核生物中，如[具体原核生物1]和[具体原核生物2]，DNA降解（Dnd）CDE蛋白复合物（由DndC、DndD和DndE组成），与DndA/IscS蛋白以及DndFGH复合物一起，作为一种防御屏障，防止非自身DNA的入侵。DndCDE复合物将硫代磷酸化（PT）修饰引入DNA，而DndFGH复合物特异性切割非PT DNA，从而限制水平基因转移和噬菌体入侵。尽管DndCDE复合物在DNA PT修饰中起着核心作用，该修饰催化DNA上的氧-硫交换，但我们对这个关键复合物的了解仍然很少。在这里，我们利用蛋白质结构预测对DndCDE复合物和一个23 bp的DNA-DndCDE复合物的结构进行了合理可靠的预测。我们发现，在DndCDE复合物的三种蛋白质中，DndC，尤其是其“特异性环”，在识别共有PT修饰序列中起关键作用。此外，发现DndD蛋白在其球状结构域上具有高度保守的结构表面，可能介导DndD以及DndCDE复合物的二聚化。此外，我们的正常模式分析表明，DndCDE复合物存在封闭状态和开放状态之间的动态转变，促进其与DNA的结合和释放。我们的结论通过使用纯化蛋白的生化分析得到了证实。总体而言，我们提供了对参与DNA硫代磷酸化的核心蛋白复合物的组装和DNA识别机制的分子见解。
