8XHS

Cryo-EM structure of free-state KmAgo

Summary for 8XHS

• Entry DOI: 10.2210/pdb8xhs/pdb
• EMDB information: 38354
• Descriptor: KmAgo (2 entities in total)
• Functional Keywords: mesophilic prokaryotic argonaute, dna binding protein
• Biological source: Kurthia massiliensis
• Total number of polymer chains: 1
• Total formula weight: 85485.20

Authors

Tao, X.,Ding, H.,Wu, S. (deposition date: 2023-12-18, release date: 2024-12-25, Last modification date: 2025-07-16)

Primary citation

Tao, X.,Ding, H.,Wu, S.,Wang, F.,Xu, H.,Li, J.,Zhai, C.,Li, S.,Chen, K.,Wu, S.,Liu, Y.,Ma, L.
Structural and mechanistic insights into a mesophilic prokaryotic Argonaute.
Nucleic Acids Res., 52:11895-11910, 2024

PubMed Abstract: 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.

PubMed: 39315697
DOI: 10.1093/nar/gkae820

Experimental method

ELECTRON MICROSCOPY (2.85 Å)