Institute of Biotechnology, Life Sciences Center, Vilnius University, Sauletekio av. 7, 10257, Vilnius, Lithuania.
Institute of Physics, Center for Physical Sciences and Technology, Savanoriu 231, 02300, Vilnius, Lithuania.
Sci Rep. 2021 Feb 25;11(1):4518. doi: 10.1038/s41598-021-83889-4.
Argonaute (Ago) proteins are found in all three domains of life. The best-characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are composed of four major structural/functional domains (N, PAZ, MID, and PIWI) and thereby closely resemble eAgos. It was demonstrated that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown. In this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including SEC-MALS, SAXS, single-molecule FRET, and AFM, to show that AfAgo is indeed a homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding underscores the diversity of prokaryotic Agos and broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.
Argonaute (Ago) 蛋白存在于生命的三个领域中。研究最深入的一组是真核 Ago（eAgos），它们是 RNA 干扰的核心。研究最透彻的原核 Ago（pAgo）蛋白是全长的 pAgos。它们由四个主要的结构/功能域（N、PAZ、MID 和 PIWI）组成，因此与 eAgos 非常相似。研究表明，全长的 pAgos 作为原核抗病毒系统发挥作用，PIWI 结构域对入侵的核酸进行切割。然而，大多数已鉴定的 pAgos 较短且无催化活性（仅编码 MID 和无活性的 PIWI 结构域），因此其作用机制和功能仍然未知。在这项工作中，我们专注于 AfAgo，一种由古菌 Archaeoglobus fulgidus 编码的短 pAgo 蛋白。我们发现，在以前解决的所有 AfAgo 结构中，其两个单体形成了涉及 C 末端β-片层的大量二聚化界面。受此发现的启发，我们采用了各种生化和生物物理测定方法，包括 SEC-MALS、SAXS、单分子 FRET 和 AFM，表明 AfAgo 在溶液中确实是同源二聚体，能够同时与两个 DNA 分子相互作用。这一发现突显了原核 Ago 的多样性，并拓宽了目前已知 Argonaute-核酸相互作用机制的范围。
