title: Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage.
abstract: Argonaute (Ago) proteins constitute a key component of the RNA-induced silencing complex (RISC). We report the crystal structure of Aquifex aeolicus Ago (Aa-Ago) together with binding and cleavage studies, which establish this eubacterial Ago as a bona fide guide DNA strand-mediated site-specific RNA endonuclease. We have generated a stereochemically robust model of the complex, where the guide DNA-mRNA duplex is positioned within a basic channel spanning the bilobal interface, such that the 5' phosphate of the guide strand can be anchored in a basic pocket, and the mRNA can be positioned for site-specific cleavage by RNase H-type divalent cation-coordinated catalytic Asp residues of the PIWI domain. Domain swap experiments involving chimeras of human Ago (hAgo1) and cleavage-competent hAgo2 reinforce the role of the PIWI domain in "slicer" activity. We propose a four-step Ago-mediated catalytic cleavage cycle model, which provides distinct perspectives into the mechanism of guide strand-mediated mRNA cleavage within the RISC.
authors.affiliation: Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
authors.forename: Yu-Ren
authors.initials: YR
authors.lastname: Yuan
authors.forename: Yi
authors.initials: Y
authors.lastname: Pei
authors.forename: Jin-Biao
authors.initials: JB
authors.lastname: Ma
authors.forename: Vitaly
authors.initials: V
authors.lastname: Kuryavyi
authors.forename: Maria
authors.initials: M
authors.lastname: Zhadina
authors.forename: Gunter
authors.initials: G
authors.lastname: Meister
authors.forename: Hong-Ying
authors.initials: HY
authors.lastname: Chen
authors.forename: Zbigniew
authors.initials: Z
authors.lastname: Dauter
authors.forename: Thomas
authors.initials: T
authors.lastname: Tuschl
authors.forename: Dinshaw J
authors.initials: DJ
authors.lastname: Patel
mesh_terms: D000595:Amino Acid Sequence; D060565:Argonaute Proteins; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D020134:Catalytic Domain / Q000235:genetics; D002413:Cations, Divalent / Q000737:chemistry; D018360:Crystallography, X-Ray; D004277:DNA, Single-Stranded / Q000378:metabolism; D004722:Endoribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000378:metabolism; D010448:Peptide Initiation Factors / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012330:RNA, Double-Stranded / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D055672:Static Electricity
chemical_list: C115848:AGO1 protein, human; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D001426:Bacterial Proteins; D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D009841:Oligonucleotides; D010448:Peptide Initiation Factors; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034743:RNA-Induced Silencing Complex; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D004722:Endoribonucleases; C504752:argonaute protein, Aquiflex aeolicus