Department of Biochemistry, School of Medicine, Southern University of Science and Technology, Shenzhen, China.
Department of Chemistry, Southern University of Science and Technology, Shenzhen, China.
Nat Commun. 2024 Oct 4;15(1):8610. doi: 10.1038/s41467-024-52910-5.
Eukaryotic Argonaute proteins (eAgos) utilize short nucleic acid guides to target complementary sequences for RNA silencing, while prokaryotic Agos (pAgos) provide immunity against invading plasmids or bacteriophages. The Sir2-domain associated short pAgo (SPARSA) immune system defends against invaders by depleting NAD and triggering cell death. However, the molecular mechanism underlying SPARSA activation remains unknown. Here, we present cryo-EM structures of inactive monomeric, active tetrameric and active NAD-bound tetrameric SPARSA complexes, elucidating mechanisms underlying SPARSA assembly, guide RNA preference, target ssDNA-triggered SPARSA tetramerization, and tetrameric-dependent NADase activation. Short pAgos form heterodimers with Sir2-APAZ, favoring short guide RNA with a 5'-AU from ColE-like plasmids. RNA-guided recognition of the target ssDNA triggers SPARSA tetramerization via pAgo- and Sir2-mediated interactions. The resulting tetrameric Sir2 rearrangement aligns catalytic residue H186 for NAD hydrolysis. These insights advance our understanding of Sir2-domain associated pAgos immune systems and should facilitate the development of a short pAgo-associated biotechnological toolbox.
真核 Argonaute 蛋白 (eAgos) 利用短核酸引导物靶向互补序列进行 RNA 沉默，而原核 Agos (pAgos) 则提供针对入侵质粒或噬菌体的免疫。Sir2 结构域相关的短 pAgo（SPARSA）免疫系统通过消耗 NAD 和触发细胞死亡来抵御入侵。然而，SPARSA 激活的分子机制仍不清楚。在这里，我们展示了无活性单体、活性四聚体和活性 NAD 结合四聚体 SPARSA 复合物的冷冻电镜结构，阐明了 SPARSA 组装、引导 RNA 偏好、靶 ssDNA 触发 SPARSA 四聚化以及四聚体依赖性 NADase 激活的机制。短 pAgos 与 Sir2-APAZ 形成异二聚体，有利于 ColE 样质粒 5'-AU 的短引导 RNA。RNA 引导的靶 ssDNA 识别触发 pAgo 和 Sir2 介导的相互作用导致 SPARSA 四聚化。由此产生的四聚体 Sir2 重排使催化残基 H186 对齐以进行 NAD 水解。这些见解增进了我们对 Sir2 结构域相关 pAgos 免疫系统的理解，并且应该有助于开发与短 pAgo 相关的生物技术工具包。
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