Nucleic acid-induced NADase activation of a short Sir2-associated prokaryotic Argonaute system

Inhibition of nucleic acid targets is mediated by Argonaute (Ago) proteins guided by RNA or DNA. Although the mechanisms underpinning the functions of eukaryotic and “long” prokaryotic Ago proteins (pAgos) are well understood, those for short pAgos remain enigmatic. Here, we determine two cryoelectron microscopy structures of short pAgos in association with the NADase-domain-containing protein Sir2-APAZ from Geobacter sulfurreducens (GsSir2/Ago): the guide RNA-target DNA-loaded GsSir2/Ago quaternary complex (2.58 Å) and the dimer of the quaternary complex (2.93Å). These structures show that the nucleic acid binding causes profound conformational changes that result in disorder or partial dissociation of the Sir2 domain, suggesting that it adopts a NADase-active conformation. Subsequently, two RNA-/DNA-loaded GsSir2/Ago complexes form a dimer through their MID domains, further enhancing NADase activity through synergistic effects. The findings provide a structural basis for short-pAgo-mediated defense against invading nucleic acids. [Display omitted] •The GsSir2-Ago is an RNA-guided DNA target NADase with canonical Ago architecture•The GsSir2-Ago monomer displays moderate NADase activity•The GsSir2-Ago dimer displays increasing NADase activity through synergistic effects Sun et al. determine the two cryo-EM structures of the monomeric GsSir2-Ago-gRNA-tDNA and dimeric GsSir2-Ago-gRNA-tDNA complexes. Structural details and mutagenesis study dissect molecular mechanisms of nucleic acid-induced NADase activation of a short Sir2-associated prokaryotic Argonaute system.