Structural and mechanistic insights into a mesophilic prokaryotic Argonaute

文献类型： 外文期刊

第一作者： Tao, Xin

作者： Tao, Xin;Ding, Hui;Wang, Fei;Xu, Hu;Li, Jie;Zhai, Chao;Li, Shunshun;Chen, Kai;Wu, Shan;Liu, Yang;Ma, Lixin;Wu, Shaowen

作者机构：

期刊名称：NUCLEIC ACIDS RESEARCH （ 影响因子：13.1； 五年影响因子：16.8 ）

ISSN： 0305-1048

年卷期： 2024 年 52 卷 19 期

页码：

收录情况： SCI

摘要： Argonaute (Ago) proteins are programmable nucleases found in all domains of life, playing a crucial role in biological processes like DNA/RNA interference and gene regulation. Mesophilic prokaryotic Agos (pAgos) have gained increasing research interest due to their broad range of potential applications, yet their molecular mechanisms remain poorly understood. Here, we present seven cryo-electron microscopy structures of Kurthia massiliensis Ago (KmAgo) in various states. These structures encompass the steps of apo-form, guide binding, target recognition, cleavage, and release, revealing that KmAgo employs a unique DDD catalytic triad, instead of a DEDD tetrad, for DNA target cleavage under 5 ' P-DNA guide conditions. Notably, the last catalytic residue, D713, is positioned outside the catalytic pocket in the absence of guide. After guide binding, D713 enters the catalytic pocket. In contrast, the corresponding catalytic residue in other Agos has been consistently located in the catalytic pocket. Moreover, we identified several sites exhibiting enhanced catalytic activity through alanine mutagenesis. These sites have the potential to serve as engineering targets for augmenting the catalytic efficiency of KmAgo. This structural analysis of KmAgo advances the understanding of the diversity of molecular mechanisms by Agos, offering insights for developing and optimizing mesophilic pAgos-based programmable DNA and RNA manipulation tools. Graphical Abstract

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