Eukaryotic Argonaute proteins induce gene silencing by small RNAguided recognition and cleavage of mRNA targets. Although structural similarities between human and prokaryotic Argonautes are consistent with shared mechanistic properties, sequence and structure-based alignments suggested that Argonautes encoded within CRISPR-cas [clustered regularly interspaced short palindromic repeats (CRISPR)-associated] bacterial immunity operons have divergent activities. We show here that the CRISPR-associated Marinitoga piezophila Argonaute (MpAgo) protein cleaves single-stranded target sequences using 5′-hydroxylated guide RNAs rather than the 5′-phosphorylated guides used by all known Argonautes. The 2.0-Å resolution crystal structure of an MpAgo-RNA complex reveals a guide strand binding site comprising residues that block 5′ phosphate interactions. Using structure-based sequence alignment, we were able to identify other putative MpAgo-like proteins, all of which are encoded within CRISPRcas loci. Taken together, our data suggest the evolution of an Argonaute subclass with noncanonical specificity for a 5′-hydroxylated guide.Argonaute | small noncoding RNA | RNA interference A rgonaute (Ago) proteins bind small RNA or DNA guides, which provide base-pairing specificity for recognition and cleavage of complementary nucleic acid targets. Members of this protein family are present in all three domains of life (1). In eukaryotes, Argonautes are the key effectors of RNA interference (RNAi) pathways that regulate posttranscriptional gene expression (2-4). However, the role of Argonaute proteins in bacteria and archaea, which lack RNAi pathways, remains poorly understood (5).Recent studies suggested that DNA-guided bacterial and archaeal Argonaute proteins are directly involved in host defense by cleaving foreign DNA elements, such as DNA viruses and plasmids (6, 7). In addition, a catalytically inactive Argonaute protein in Rhodobacter sphaeroides (RsAgo) was demonstrated to use RNA guides and possibly recruits an associated nuclease for subsequent target cleavage (8). Despite these divergent modes of action, bacterial and archaeal Argonaute proteins adopt a highly conserved bilobed architecture. Herein, an N-terminal and a PIWI-ArgonauteZwille (PAZ) domain constitute one lobe, whereas the other lobe consists of the middle (MID) domain and the catalytic RNase H-like P element-induced wimpy testis (PIWI) domain (9-15). Molecular structures of a eukaryotic Argonaute MID domain and an Archaeoglobus fulgidus Piwi (AfPiwi) enzyme bound to a guide RNA showed the importance of the 5′-terminal base identity, as well as the 5′ phosphate in guide strand binding, to Ago (10,[13][14][15][16][17]. Notably, recognition of the 5′ end of the guide in the MID domain and guide strand preorganization for target interaction are conserved across the entire Argonaute superfamily (1).The nucleic acid-guided binding and cleavage activities of Argonaute proteins are reminiscent of the activities of RNA-guided proteins within CRISPR-Cas systems [cluste...