Structural and mechanistic insights into a mesophilic prokaryotic Argonaute

作者：Xin Tao, Hui Ding, Shaowen Wu, Shaowen Wu, Fei Wang, Hu Xu, Jie Li, Chao Zhai, Shunshun Li, Kai Chen, Shan Wu, Shan Wu, Yang Liu, Lixin Ma · 发表于：Nucleic Acids Research · 年份：2024 · DOI：10.1093/nar/gkae820 · 被引用次数：12 · 研究领域：Advanced biosensing and bioanalysis techniques、RNA and protein synthesis mechanisms、CRISPR and Genetic Engineering

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.