State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, Hubei 430062, China.
State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, Hubei 430062, China.
Mol Cell. 2024 Feb 15;84(4):675-686.e4. doi: 10.1016/j.molcel.2024.01.004. Epub 2024 Jan 30.
The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo-guide DNA-target DNA ternary complex at the cleavage-compatible state. The ternary complex is predominantly dimerized, and the dimerization is solely mediated by PfAgo at PIWI-MID, PIWI-PIWI, and PAZ-N interfaces. Additionally, PfAgo accommodates a short 14-bp guide-target DNA duplex with a wedge-type N domain and specifically recognizes 5'-phosphorylated guide DNA. In contrast, the PfAgo-guide DNA binary complex is monomeric, and the engagement of target DNA with 14-bp complementarity induces sufficient dimerization and activation of PfAgo, accompanied by movement of PAZ and N domains. A closely related Argonaute from Thermococcus thioreducens adopts a similar dimerization configuration with an additional zinc finger formed at the dimerization interface. Dimerization of both Argonautes stabilizes the catalytic loops, highlighting the important role of Argonaute dimerization in the activation and target cleavage.
来自嗜热古菌 Pyrococcus furiosus（PfAgo）的 Argonaute 核酸内切酶有助于宿主防御，是一种很有前途的生物技术工具。在此，我们报告了 PfAgo-引导 DNA-靶 DNA 三元复合物在可切割状态下的结构。三元复合物主要以二聚体形式存在，二聚体仅通过 PfAgo 在 PIWI-MID、PIWI-PIWI 和 PAZ-N 界面介导。此外，PfAgo 可容纳带有楔形 N 结构域的短 14 个碱基对的引导物-靶 DNA 双链体，并特异性识别 5'-磷酸化的引导 DNA。相比之下，PfAgo-引导 DNA 二元复合物是单体的，与具有 14 个碱基互补的靶 DNA 的结合诱导足够的二聚化和 PfAgo 的激活，同时 PAZ 和 N 结构域发生移动。来自 Thermococcus thioreducens 的密切相关的 Argonaute 采用类似的二聚化构象，在二聚化界面形成额外的锌指。这两种 Argonaute 的二聚化稳定了催化环，突出了 Argonaute 二聚化在激活和靶切割中的重要作用。
