Work

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

Year: 2026

Type: article

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.

Source: Nucleic Acids Research

DOI: 10.1093/nar/gkag1001

Authors: Jin Zhang, Zengru Li, Le Ye, Yaoyao Hou, Ke Liu, Jinrong Min

Institutions: Central China Normal University, Huazhong University of Science and Technology

Language: English

Topic: CRISPR and Genetic Engineering

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $3,992

Funders: National Natural Science Foundation of China, Central China Normal University