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Peer-reviewedCC BY-NC 4.0Nucleic Acids Research

Structural basis of assembly-dependent activation of the MAD3–MAD4 nuclease in the Pseudomonas aeruginosa MADS defense system

  • Jin Zhang1
  • Zengru Li2,3
  • Le Ye1
  • Yaoyao Hou1
  • Ke Liu1
  • Jinrong Min1
  1. 1Key Laboratory of Pesticide & Chemical Biology, Ministry of Education, Hubei Key Laboratory of Genetic Regulation and Integrative Biology, School of Life Sciences, Central China Normal University , Wuhan 430079 ,
  2. 2Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology , Wuhan 430074 ,
  3. 3Center for Biological Cryo-EM, Huazhong University of Science and Technology , Wuhan 430075 ,
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Abstract

The methylation-associated defense system (MADS) is a bacterial antiphage immune system that cooperates with CRISPR–Cas systems to restrict phage infection. Within MADS, the MAD3–MAD4 module has been proposed to function as an ATPase–nuclease module related to the OLD nuclease family, but the molecular basis of its assembly and function remains unknown. Here, we combined cryo-electron microscopy, X-ray crystallography, biochemical assays, and mutagenesis to elucidate the architecture and assembly mechanism of the MAD3–MAD4 complex. We found that MAD3 and MAD4 assemble into a stable 2:2 heterotetramer. Structural and biochemical analyses revealed that MAD3 alone lacks detectable ATPase activity, whereas MAD4 is insoluble when expressed alone. Assembly of the MAD3–MAD4 complex induces a conformational rearrangement in which MAD3 adopts an ATPase-competent dimeric conformation, while MAD4 is stabilized through intersubunit interactions. This heterotetrameric assembly exhibits both ATPase and DNA cleavage activities. Overall, our findings reveal the molecular basis of MAD3–MAD4 heterotetramer assembly and provide the first structural and biochemical insights into the function of the MAD3–MAD4 module within the MADS defense system.

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Cite

BibTeXRIS
BibTeX
@article{zhang2026structural,
  title = {{Structural basis of assembly-dependent activation of the MAD3–MAD4 nuclease in the Pseudomonas aeruginosa MADS defense system}},
  author = {Jin Zhang and Zengru Li and Le Ye and Yaoyao Hou and Ke Liu and Jinrong Min},
  journal = {Nucleic Acids Research},
  year = {2026},
  month = oct,
  doi = {10.1093/nar/gkag1001},
  url = {https://academic.oup.com/nar/article/doi/10.1093/nar/gkag1001/8915705},
  note = {CC BY-NC 4.0},
}
RIS
TY  - JOUR
TI  - Structural basis of assembly-dependent activation of the MAD3–MAD4 nuclease in the Pseudomonas aeruginosa MADS defense system
AU  - Jin Zhang
AU  - Zengru Li
AU  - Le Ye
AU  - Yaoyao Hou
AU  - Ke Liu
AU  - Jinrong Min
PY  - 2026
DA  - 2026/10/14
JO  - Nucleic Acids Research
DO  - 10.1093/nar/gkag1001
UR  - https://academic.oup.com/nar/article/doi/10.1093/nar/gkag1001/8915705
AB  - The methylation-associated defense system (MADS) is a bacterial antiphage immune system that cooperates with CRISPR–Cas systems to restrict phage infection. Within MADS, the MAD3–MAD4 module has been proposed to function as an ATPase–nuclease module related to the OLD nuclease family, but the molecular basis of its assembly and function remains unknown. Here, we combined cryo-electron microscopy, X-ray crystallography, biochemical assays, and mutagenesis to elucidate the architecture and assembly mechanism of the MAD3–MAD4 complex. We found that MAD3 and MAD4 assemble into a stable 2:2 heterotetramer. Structural and biochemical analyses revealed that MAD3 alone lacks detectable ATPase activity, whereas MAD4 is insoluble when expressed alone. Assembly of the MAD3–MAD4 complex induces a conformational rearrangement in which MAD3 adopts an ATPase-competent dimeric conformation, while MAD4 is stabilized through intersubunit interactions. This heterotetrameric assembly exhibits both ATPase and DNA cleavage activities. Overall, our findings reveal the molecular basis of MAD3–MAD4 heterotetramer assembly and provide the first structural and biochemical insights into the function of the MAD3–MAD4 module within the MADS defense system.
SN  - 0305-1048
SN  - 1362-4962
ER  -