Crystal structure of A. aeolicus argonaute, a site-specific DNA-guided endoribonuclease, provides insights into RISC-mediated mRNA cleavage.
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.
Yuan
Yu-Ren
YR
Structural Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Pei
Yi
Y
Ma
Jin-Biao
JB
Kuryavyi
Vitaly
V
Zhadina
Maria
M
Meister
Gunter
G
Chen
Hong-Ying
HY
Dauter
Zbigniew
Z
Tuschl
Thomas
T
Patel
Dinshaw J
DJ
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
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