Structural and mechanistic insights into a mesophilic prokaryotic Argonaute.

Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE

Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.

Argonaute Proteins*/metabolism ; Argonaute Proteins*/chemistry ; Argonaute Proteins*/genetics ; Cryoelectron Microscopy* ; Models, Molecular*; Catalytic Domain ; DNA/chemistry ; DNA/metabolism ; Protein Binding ; Bacterial Proteins/chemistry ; Bacterial Proteins/metabolism ; Bacterial Proteins/genetics

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.
(© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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2023YFA0913700 National Key Research and Development Program of China; 32300517 National Natural Science Foundation of China; 2022M721074 China Postdoctoral Science Foundation; 2022CFA078 Distinguished Young Scholars of Hubei Province; 2023020201020418 Knowledge Innovation Program of Wuhan-Shuguang Project

0 (Argonaute Proteins)
9007-49-2 (DNA)
0 (Bacterial Proteins)

Date Created: 20240924 Date Completed: 20241029 Latest Revision: 20241030

20250114

PMC11514475

10.1093/nar/gkae820

39315697