Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein Andreas Boland, Eric Huntzinger, Steffen Schmidt, Elisa Izaurralde1, and Oliver Weichenrieder1 Max Planck Institute for Developmental Biology, Spemannstrasse 35, D-72076 Tübingen, Germany Edited by Jennifer A. Doudna, University of California, Berkeley, CA, and approved May 6, 2011 (received for review March 10, 2011) Argonaute proteins (AGOs) are essential effectors in RNA- involved in the micro-RNA (miRNA) pathway] contain a second, mediated gene silencing pathways. They are characterized by a allosteric nucleotide binding site with an affinity for m7GpppG bilobal architecture, in which one lobe contains the N-terminal cap analogs. However, considering the structures of the prokaryotic and PAZ domains and the other contains the MID and PIWI do- AGOs, it was unclear whether the putative second ligand-binding mains. Here, we present the first crystal structure of the MID-PIWI site would be accessible in the presence of the PIWI domain. lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein. Toaddress this question and gain further molecular insight into Compared to prokaryotic AGOs, the domain orientation is con- eukaryotic AGOs, we determined the crystal structure of the served, indicating a conserved mode of nucleic acid binding. The entire MID-PIWI lobe of the Nc QDE-2 protein, tested its PIWI domain shows an adaptable surface loop next to a eukar- RNA-binding properties in vitro, and analyzed its implications yote-specific α-helical insertion, which are both likely to contact in vivo in the context of the Dm AGO1 protein. The structure the PAZ domain in a conformation-dependent manner to sense the provides a detailed, high-resolution view of the MID-PIWI inter- functional state of the protein. The MID-PIWI interface is hydrophi- face in a eukaryotic AGO protein and shows that the two domains lic and buries residues that were previously thought to participate are oriented very similarly to their prokaryotic counterparts, directly in the allosteric regulation of guide RNA binding. The inter- indicating a conserved mode of guide RNA/DNA strand recogni- face includes the binding pocket for the guide RNA 5′ end, and tion. However, despite these similarities, the PIWI-domain residues from both domains contribute to binding. Accordingly, exhibits eukaryote-specific structural features that might act as micro-RNA (miRNA) binding is particularly sensitive to alteration in sensors for the functional state of the protein. Finally, we show the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. that residues that have been implicated in allosteric regulation in The structure of the QDE-2 MID-PIWI lobe provides molecular and previous studies (21) are in fact involved in MID-PIWI interdo- mechanistic insight into eukaryotic AGOs and has significant impli- main interactions and contribute to guide RNA binding by cations for understanding the role of these proteins in silencing. stabilizing the MID-PIWI interface. roteins of the Argonaute (AGO) family play essential roles in Results PRNA-mediated gene silencing mechanisms in eukaryotes The Eukaryotic AGO MID-PIWI Lobe Adopts a Fold Highly Similar to the (1, 2). They are loaded with small noncoding RNAs to form the Prokaryotic Homologs. The Neurospora crassa Argonaute protein core of RNA-induced silencing complexes, which repress the QDE-2 (Nc QDE-2) is a close sequence homolog of eukaryotic expression of target genes at the transcriptional or posttran- AGOs that act in the small interfering RNA (siRNA) and miR- scriptional level (1, 2). The targets to be silenced are selected NA pathways [e.g., sequence identities with Hs AGO2 and Dm through base-pairing interactions between the loaded small RNA AGO1 are 30% and 29.7%, respectively (Fig. S1) (22, 23)]. We (also known as the guide RNA) and an mRNA target containing obtained diffracting crystals of a QDE-2 fragment containing the partially or fully complementary sequences (1–3). MID and PIWI domains (amino acids 506–938) and determined Thus far, structural information on full-length AGOs has been the structure at 3.65 Å resolution (crystal form I; Table S1). The available only for the homologous proteins from Archaea and structure revealed a disordered loop (loop L3, Fig. 1A, and Eubacteria, which preferentially use DNA as a guide (4–10). Fig. S1) including amino acids K786–A840 of the PIWI domain, These studies revealed that AGOs consist of four domains: the which we replaced with a Gly-Ser (GSG) linker to generate crys- N-terminal domain; the PAZ domain, which binds the 3′ end tals of a MID-PIWI ΔL3 protein that diffracted to 1.85 Å resolu- R of guide RNAs/DNAs; the MID domain, which provides a bind- tion. This structure (crystal form II) was refined to an work of ′ R ¼ 23 6% R ing pocket for the 5 phosphate of guide RNAs/DNAs; and the 19.6% ( free . ), whereas crystal form I yielded an work R ¼ 25 2% PIWI domain, which adopts an RNase H fold and has endonu- of 23.3% ( free . )(Table S1). cleolytic activity in some, but not all, AGOs (4–11). This structure reveals the details of a eukaryotic AGO MID- For the eukaryotic AGO clade of Argonaute proteins, structural PIWI lobe. The individual MID and PIWI domains superimpose information is available only for the isolated PAZ domains of well with the structures of previously determined archaeal and Drosophila melanogaster (Dm) AGO1 and AGO2, human AGO1 eubacterial AGO MID and PIWI domains (Fig. 1A vs. 1B and (12–16) and the MID domains of human AGO2 and Neurospora crassa (Nc) QDE-2 (17, 18). Structural information is also avail- able for PAZ domains of the PIWI clade of AGOs (19, 20). These Author contributions: A.B., E.I., and O.W. designed research; A.B., E.H., and O.W. performed research; A.B., E.H., S.S., E.I., and O.W. analyzed data; and A.B., E.I., and studies showed that the PAZ and MID domains of eukaryotic O.W. wrote the paper. AGOs adopt folds similar to the prokaryotic homologs and recog- The authors declare no conflict of interest. nize the 3′-and5′-terminal nucleotides of the guide strand, respec- This article is a PNAS Direct Submission. tively, in a similar manner to their prokaryotic counterparts (12–18). Our previous structure of the isolated Nc QDE-2 MID domain Freely available online through the PNAS open access option. revealed that the 5′-nucleotide binding site shares residues with a Data deposition: The accession codes and coordinates of the QDE-2 MID-PIWI lobe and ′ MID-PIWI ΔL3 have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes second, adjacent sulfate ion-binding site, suggesting that the 5 - 2yhb and 2yha). terminal nucleotide of the guide RNA and a second ligand may bind 1To whom correspondence may be addressed. E-mail: [email protected] cooperatively to the MID domain of eukaryotic AGOs (17). These or [email protected]. findings supported the observation of Djuranovic et al. (21) that This article contains supporting information online at www.pnas.org/lookup/suppl/ the isolated MID domains of certain eukaryotic AGOs [i.e., those doi:10.1073/pnas.1103946108/-/DCSupplemental. 10466–10471 ∣ PNAS ∣ June 28, 2011 ∣ vol. 108 ∣ no. 26 www.pnas.org/cgi/doi/10.1073/pnas.1103946108 Downloaded by guest on September 26, 2021 here to the AGO clade, which contains Nc QDE-2 and eukaryotic AGOs involved in the siRNA and miRNA pathways. The PIWI Domain and the Interaction with Target mRNA. The PIWI domain of Nc QDE-2 (residues H644–I938) adopts an RNase H fold with a catalytically active DDD motif, which is less common than the DDH motif present in most eukaryotic AGOs (Fig. S1). A structure-based alignment (Fig. S1) identified a series of loops on the putative nucleic acid-binding surface of the PIWI domain [Fig. 1A, loops L1 (H667–P681), L2 (G746–Q751), L3 (K788– A840), and L4 (F873–I882)]. Their significance is best under- stood in the context of a double stranded nucleic acid substrate that can be placed on the QDE-2 MID-PIWI lobe by the struc- tural superposition with Thermus thermophilus AGO-nucleic acid complex [Fig. 1 B and C; Protein Data Bank (PDB) ID code 3HJF; ref. 10]. In this particular model substrate, the 3′ end of the guide DNA strand is not anchored in the PAZ domain. Instead, nucleotides 2 to 15 of the guide DNA are base-paired to an RNA target strand, forming a duplex extending beyond the seed sequence (see Fig. 1D for a numbering of the respective nucleotides), which is typical for siRNA targets but rather rare for animal miRNA targets (3). The scissile bond of the model target strand (between nucleo- tides 10′ and 11′) fits nicely in the catalytic site (Fig. 2 A and B), and loop L2 is perfectly positioned to probe the minor groove of the duplex (base pair 13) with S748 (Fig. 2 C and D). Loop L2 is highly conserved in all eukaryotic AGOs and likely fixes the phos- phodiester backbone of the target strand (nucleotides 12′ and 13′) via E749 and nucleotide 14 from the guide strand via Q751 (Fig. 2 C and D) in cases where the downstream duplex is formed (e.g., in the case of fully complementary targets). Loop L3 is disordered in crystal form I and was deleted in crys- tal form II. Judging from the prokaryotic AGO structures, the central parts of this loop likely organize the missing lobe (contain- ing the N-term and PAZ domains) of the AGO protein (Fig. S2B), whereas the N-terminal residues of loop L3 [including the con- served K786 (Fig.