Mechanism of the long-B prokaryotic Argonaute-mediated nuclease activation in bacterial immunity

Prokaryotic Argonautes (pAgos) are widely distributed and provide immunity against invading DNA. Based on their domain architecture, pAgos are classified into three major groups: long-A, long-B, and short pAgos. Among them, long-B pAgos remain the least understood subgroup. Here, we show that a long-B pAgo-nuclease system (EcBPAN) from Escherichia coli (E. coli) provides the first clear evidence of anti-phage activity among long-B pAgo systems. By combining structural determination, biochemical analyses, and in vivo phage-resistance assays, we elucidated the activation mechanism of EcBPAN. We found that RNA-guided EcAgo recognizes target DNA and subsequently recruits the autoinhibited EcbAgaN (long-B pAgo-associated nuclease from E. coli) dimer to form an unprecedented 8:8 pAgo-nuclease complex with robust nonspecific DNase activity. The cryo-EM structure of the activated complex revealed a distinctive bowl-shaped architecture, in which the C-terminal nuclease domains of EcbAgaN form an active octamer, while the N-terminal domains engage four EcAgo-guide RNA-target DNA ternary dimers in an interleaved manner, thereby relieving autoinhibition. Together, these findings provide mechanistic insights into the long-B pAgo defense system and reveal a mode of immune activation that is distinct from those of long-A and short pAgo systems, its supramolecular assembly and regulatory mechanism.