Mechanistic Insights into Cyclic Penta-Adenylate-Mediated Activation of Type III CRISPR Ribonuclease Csm6

Type III CRISPR systems generate cyclic oligoadenylate (cOA, 3 to 6 AMPs) messengers upon detecting viral RNA, activating downstream effectors to defend against viral infection. Although cOA-activated effectors have been extensively characterized, the cA ₅ -specific effectors remained unexplored despite cA ₅ being among the most abundant cOA species produced during phage infection. Here, we report that Actinomyces procaprae Csm6 (ApCsm6) selectively employs cA ₅ as its activator. Unlike other characterized Csm6 proteins, ApCsm6 self-limits its ribonuclease activity by degrading cOAs via its HEPN domain, rather than relaying on the CARF domain. Cryo-EM structures of ApCsm6 and its complexes with cA ₅ and cA ₆ reveal a homotetrameric assembly, where each monomer binds a single cOA within a composite pocket formed by two tandem CARF-HEPN domains. Binding of cA ₅ , but not cA ₆ , enhances tetramerization and induces large conformational shifts in CARF, which in turn allosterically activates ssRNA cleavage in HEPN. These findings advance our understanding of ligand discrimination and signaling regulation in type III CRISPR immunity.