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PreprintCited by 11CRISPR and Genetic EngineeringDNA and Nucleic Acid ChemistryRNA regulation and disease

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Mn 2+ -Induced Structural Flexibility Enhances the Entire Catalytic Cycle and the Cleavage of Mismatches in Prokaryotic Argonaute Proteins

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Zheng, Lirong, Zan, Bing, Yang, Yu, Zhou, Bingxin, Wu, Banghao, Feng, Yan, Liu, Qian, Hong, Liang·2023·Chemical Science
DOI 10.1101/2023.06.28.546964PMC PMC11023060

Preprint — published as 10.1039/d3sc06221j · Paper page

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Abstract

Prokaryotic Argonaute (pAgo) proteins, a class of DNA/RNA-guided programmable endonucleases, have been extensively utilized in nucleic acid biosensors. The specific binding and cleavage of nucleic acids by pAgo proteins, which are crucial processes for their applications, are dependent on the presence of Mn 2+ bound in the pockets, as verified through X-ray crystallography. However, a comprehensive understanding of how dissociated Mn 2+ in the solvent affects the catalytic cycle, and its underlying regulatory role in this structure-function relationship, remains underdetermined. By combining experimental and computational methods, this study reveals that unbound Mn 2+ in solution enhances the flexibility of diverse pAgo proteins. This increase in flexibility through decreasing the number of hydrogen bonds, induced by Mn 2+ , leads to higher affinity for substrates, thus facilitating cleavage. More importantly, Mn 2+ -induced structural flexibility increases the mismatch tolerance between guide-target pairs by increasing the conformational states, thereby enhancing the cleavage of mismatches. Further simulations indicated that the enhanced flexibility in linkers triggers conformational changes in the PAZ domain for recognizing various lengths of nucleic acids. Additionally, Mn 2+ -induced dynamic alterations of the protein cause a conformational shift in the N domain and catalytic sites towards their functional form, resulting in a decreased energy penalty for target release and cleavage. These findings demonstrate that the dynamic conformations of pAgo proteins, resulting from the presence of the unbound Mn 2+ in solution, significantly promote the catalytic cycle of endonucleases and the tolerance of cleavage to mismatches. This flexibility enhancement mechanism serves as a general strategy employed by Ago proteins from diverse prokaryotes to accomplish their catalytic functions and provide useful information for Ago-based precise molecular diagnostics.

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Metadata

Authors
Zheng, L., Zan, B., Yang, Y., Zhou, B., Wu, B., Feng, Y., Liu, Q., & Hong, L.
Affiliations
Shanghai Jiao Tong University (CN)
Corresponding author
  • Zhou, Bingxin
  • Liu, Qian
  • Hong, Liang
ORCID
Venue
Chemical Science
Publisher
openRxiv
Published
2023-06-29
Volume
15
Issue
15
Pages
5612-5626
ISSN
2041-6520 (ISSN-L)
DOI
10.1101/2023.06.28.546964
PMC
PMC11023060
OpenAlex
W4392807531
Cited by
11
References
1
Citations by year
2026: 6 · 2025: 3 · 2024: 2
FWCI
1.49
Type
journal article
Language
en
Open access
Licence
cc-by-nc
Locations
  • Chemical Science (publishedVersion, cc-by-nc)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • National Science Foundation
  • Salt Science Research Foundation
  • National Natural Science Foundation of China (22063007, 62302291, 11974239)
  • Shanghai Municipal Education Commission
  • Shanghai Jiao Tong University (21TQ1400204)
  • National Key Research and Development Program of China (2018YFA0900403)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA regulation and disease
Keywords
Argonaute proteins, prokaryotic Argonaute, manganese(II), structural flexibility, mismatch tolerance, RNA cleavage

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