Key Laboratory of Microbial Pathogenesis and Interventions of Fujian Province University, the Key Laboratory of Innate Immune Biology of Fujian Province, Biomedical Research Center of South China, Key Laboratory of OptoElectronic Science and Technology for Medicine of the Ministry of Education, College of Life Sciences, Fujian Normal University, Fuzhou, 350117, China.
Key Laboratory of Combinatorial Biosynthesis and Drug Discovery (Ministry of Education), School of Pharmaceutical Sciences, Department of Neurosurgery, Zhongnan Hospital of Wuhan University, Wuhan, China Wuhan University, Wuhan, 430071, China.
Nat Commun. 2024 Jan 11;15(1):450. doi: 10.1038/s41467-023-44660-7.
Argonaute (Ago) proteins are ubiquitous across all kingdoms of life. Eukaryotic Agos (eAgos) use small RNAs to recognize transcripts for RNA silencing in eukaryotes. In contrast, the functions of prokaryotic counterparts (pAgo) are less well known. Recently, short pAgos in conjunction with the associated TIR or Sir2 (SPARTA or SPARSA) were found to serve as antiviral systems to combat phage infections. Herein, we present the cryo-EM structures of nicotinamide adenine dinucleotide (NAD)-bound SPARSA with and without nucleic acids at resolutions of 3.1 Å and 3.6 Å, respectively. Our results reveal that the APAZ (Analogue of PAZ) domain and the short pAgo form a featured architecture similar to the long pAgo to accommodate nucleic acids. We further identified the key residues for NAD binding and elucidated the structural basis for guide RNA and target DNA recognition. Using structural comparisons, molecular dynamics simulations, and biochemical experiments, we proposed a putative mechanism for NAD hydrolysis in which an H186 loop mediates nucleophilic attack by catalytic water molecules. Overall, our study provides mechanistic insight into the antiphage role of the SPARSA system.
Argonaute (Ago) 蛋白在所有生命领域中都普遍存在。真核 Ago（eAgos）利用小 RNA 识别真核生物中的 RNA 沉默转录物。相比之下，原核对应物（pAgo）的功能知之甚少。最近，短 pAgo 与相关的 TIR 或 Sir2（SPARTA 或 SPARSA）一起被发现作为抗病毒系统，以对抗噬菌体感染。在此，我们展示了结合和不结合核酸的烟酰胺腺嘌呤二核苷酸（NAD）结合 SPARSA 的冷冻电镜结构，分辨率分别为 3.1Å 和 3.6Å。我们的结果表明，APAZ（PAZ 类似物）结构域和短 pAgo 形成了一个独特的结构，类似于长 pAgo，以容纳核酸。我们进一步确定了 NAD 结合的关键残基，并阐明了指导 RNA 和靶 DNA 识别的结构基础。通过结构比较、分子动力学模拟和生化实验，我们提出了 NAD 水解的可能机制，其中 H186 环介导催化水分子的亲核攻击。总的来说，我们的研究为 SPARSA 系统的抗噬菌体作用提供了机制上的见解。
