Bacterial Shedu immune nucleases share a common enzymatic core regulated by diverse sensor domains.

Prokaryotes possess diverse anti-bacteriophage immune systems, including the single-protein Shedu nuclease. Here, we reveal the structural basis for activation of Bacillus cereus Shedu. Two cryoelectron microscopy structures of Shedu show that it switches between inactive and active states through conformational changes affecting active-site architecture, which are controlled by the protein's N-terminal domain (NTD). We find that B. cereus Shedu cleaves near DNA ends with a 3' single-stranded overhang, likely enabling it to specifically degrade the DNA injected by certain bacteriophages. Bioinformatic analysis of Shedu homologs reveals a conserved nuclease domain with remarkably diverse N-terminal regulatory domains: we identify 79 distinct NTD types falling into eight broad classes, including those with predicted nucleic acid binding, enzymatic, and other activities. Together, these data reveal Shedu as a broad family of immune nucleases with a common nuclease core regulated by diverse NTDs that likely respond to a range of signals.