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

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.