Structure of Aquifex aeolicus Argonaute Highlights Conformational Flexibility of the PAZ Domain as a Potential Regulator of RNA-induced Silencing Complex Function

Abstract

Gene silencing mediated by RNA interference requires the sequence-specific recognition of target mRNA by the endonuclease Argonaute, the primary enzymatic component of the RNA-induced silencing complex. We report the crystal structure of Aquifex aeolicus Argonaute, refined at 3.2 Å resolution. Relative to recent Argonaute structures, a 24°reorientation of the PAZ domain in our structure opens a basic cleft between the N-terminal and PAZ domains, exposing the guide strand binding pocket of PAZ. This rearrangement leads to a branched, Y-shaped system of grooves that extends through the molecule and merges in a central channel containing the catalytic residues. A 5.5-ns molecular dynamics simulation of Argonaute shows a strong tendency of the PAZ and N-terminal domains to be mobile. Binding of single-stranded DNA to Argonaute monitored by total internal reflection fluorescence spectroscopy shows biphasic kinetics, also indicative of domain rearrangement upon DNA binding. Conformational rearrangement of the PAZ domain may therefore be critical for the catalytic cycle of Argonaute and the RNA-induced silencing complex.RNA interference, an ancient mechanism of gene silencing mediated by small RNAs, is widely distributed throughout most eukaryotes (1, 2). Potent silencing of genes is achieved through the processing of double-stranded RNA (dsRNA) 2 into small interfering RNAs (siRNA) by the enzyme Dicer. The ϳ22-ntlong siRNAs contain a signature 5Ј-phosphate group and a 2-nt-long 3Ј-overhang (3). The siRNA is then loaded into the RNA-induced silencing complex (RISC), of which Argonaute is the primary catalytic component (4, 5). Energetic asymmetry of the siRNA ends allows for its directional loading into RISC (6 -8). Argonaute cleaves the passenger strand of the siRNA, leaving the guide strand of the siRNA bound to RISC (9 -11). This single-stranded guide strand siRNA bound to Argonaute is required for the sequence-dependent recognition of mRNA by RISC. Recombinantly expressed human Argonaute 2 in complex with single-stranded siRNA forms a minimal RISC, capable of mRNA recognition and cleavage (12). Recent crystal structures of Argonaute apoenzymes show the architecture of Argonaute to be a multidomain protein composed of N-terminal, PAZ, MID, and PIWI domains (13,14).The catalytic cycle of RISC requires the binding of a number of different nucleotide structures to Argonaute, and we expect Argonaute to undergo a number of conformational changes during the cycle of mRNA recognition by RISC (15, 16). Nevertheless, it remains unclear how the multidomain arrangement of Argonaute recognizes and distinguishes between singlestranded and double-stranded oligonucleotides that correspond to the Dicer-processed siRNA product, guide strand siRNA, and the guide strand/mRNA duplex.We report the crystal structure of Aquifex aeolicus Argonaute (AaAgo), crystallized in the presence of a single-stranded siRNA and refined using data to 3.2 Å resolution. We propose a model for Argonaute activity in the context of the RISC complex. We also ...

Nucleotide bias of DCL and AGO in plant anti-virus gene silencing

“…2 and 3 showed that G was preferred over C at most middle positions in vsiRNA asymmetry. This seems to support a hypothesis that flexibility of the RNA binding PAZ domain may contribute to recognition of siRNA in "initial loading" to the AGO complex (Rashid et al, 2007) and further suggests that the PAZ domain of plant AGO may operate a generic preference of G over C. Alternatively, the PIWI domain (Ma et al, 2005;Parker et al, 2005) may mediate unknown catalytic bias that leads to survival of the G-rich strand. Fig.…”

Single-molecule FRET supports the two-state model of Argonaute action

“…S10). 11,33 We were able to follow ternary complex formation in gel shift experiments, which showed that MjAgo efficiently binds DNA/DNA and DNA/RNA hybrids. Target strand incorporation was not observed if a DNA/DNA hybrid was used that carried a tandem mismatch as common in miRNAs that are recognized by human Ago2.…”

“…The first Ago structure published was that of an archaeal homolog isolated from Pyrococcus furiosus. 8 Since then, structures of Ago derived from bacterial, 11,12 archaeal, 8,13 and recently from eukaryotic sources [14][15][16] have been solved and show a high degree of similarity. 17,18 The protein is composed of an N-terminal domain, a PAZ, middle (MID), and PIWI domain (Fig.…”

“…21 In the ternary complex, Ago adopts a more open conformation in which the N-terminal and PAZ domains are rotated away from the MID-PIWI domains to accommodate the target RNA. 22,23 The rotation of the PAZ domain leads to the release of the 3′ end of the guide strand from the PAZ domain, which in turn allows the correct positioning of the cleavage site in the RNA toward the catalytic residues in the PIWI domain. Despite those structural insights, a description of the dynamic behavior of the Ago PAZ-domain and the loaded nucleic acid strands remains elusive.…”

A bacterial Argonaute with noncanonical guide RNA specificity

“…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)(10)(11)(12)(13)(14)(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).…”