Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage

Significance

We have solved crystal structures of ternary Thermus thermophilus Argonaute (Ago) complexes with guide and target DNA in cleavage-incompatible, cleavage-compatible, and postcleavage states in the 2.2- to 2.3-Å resolution range, thereby identifying the relative positions of catalytic residues, a pair of Mg²⁺ cations, and the nucleophilic water poised for in-line attack on the cleavable phosphate. These higher resolution structures represent snapshots of distinct key steps in the catalytic RNase H-mediated cleavage pathway, providing additional detailed insights into Ago-mediated cleavage chemistry of target strands. Importantly, a Glu residue shifts from an “outside” to an “inside” conformation where it inserts into the catalytic pocket to complete a catalytic tetrad during the transition from a cleavage-incompatible to a cleavage-compatible conformation.

Abstract

We report on crystal structures of ternary Thermus thermophilus Argonaute (TtAgo) complexes with 5′-phosphorylated guide DNA and a series of DNA targets. These ternary complex structures of cleavage-incompatible, cleavage-compatible, and postcleavage states solved at improved resolution up to 2.2 Å have provided molecular insights into the orchestrated positioning of catalytic residues, a pair of Mg²⁺ cations, and the putative water nucleophile positioned for in-line attack on the cleavable phosphate for TtAgo-mediated target cleavage by a RNase H-type mechanism. In addition, these ternary complex structures have provided insights into protein and DNA conformational changes that facilitate transition between cleavage-incompatible and cleavage-compatible states, including the role of a Glu finger in generating a cleavage-competent catalytic Asp-Glu-Asp-Asp tetrad. Following cleavage, the seed segment forms a stable duplex with the complementary segment of the target strand.

Argonaute (Ago) proteins, critical components of the RNA-induced silencing complex, play a key role in guide strand-mediated target RNA recognition, cleavage, and product release (reviewed in refs. 1–3). Ago proteins adopt a bilobal scaffold composed of an amino terminal PAZ-containing lobe (N and PAZ domains), a carboxyl-terminal PIWI-containing lobe (Mid and PIWI domains), and connecting linkers L1 and L2. Ago proteins bind guide strands whose 5′-phosphorylated and 3′-hydroxyl ends are anchored within Mid and PAZ pockets, respectively (4–7), with the anchored guide strand then serving as a template for pairing with the target strand (8, 9). The cleavage activity of Ago resides in the RNase H fold adopted by the PIWI domain (10, 11), whereby the enzyme’s Asp-Asp-Asp/His catalytic triad (12–15) initially processes loaded double-stranded siRNAs by cleaving the passenger strand and subsequently processes guide-target RNA duplexes by cleaving the target strand (reviewed in refs. 16–18). Such Mg²⁺ cation-mediated endonucleolytic cleavage of the target RNA strand (19, 20) resulting in 3′-OH and 5′-phosphate ends (21) requires Watson–Crick pairing of the guide and target strands spanning the seed segment (positions 2–2′ to 8–8′) and the cleavage site (10′–11′ step on the target strand) (9). Insights into target RNA recognition and cleavage have emerged from structural (9), chemical (22), and biophysical (23) experiments.

Notably, bacterial and archaeal Ago proteins have recently been shown to preferentially bind 5′-phosphoryated guide DNA (14, 15) and use an activated water molecule as the nucleophile (reviewed in ref. 24) to cleave both RNA and DNA target strands (9). Structural studies have been undertaken on bacterial and archaeal Ago proteins in the free state (10, 15) and bound to a 5′-phosphorylated guide DNA strand (4) and added target RNA strand (8, 9). The structural studies of Thermus thermophilus Ago (TtAgo) ternary complexes have provided insights into the nucleation, propagation, and cleavage steps of target RNA silencing in a bacterial system (9). These studies have highlighted the conformational transitions on proceeding from Ago in the free state to the binary complex (4) to the ternary complexes (8, 9) and have emphasized the requirement for a precisely aligned Asp-Asp-Asp triad and a pair of Mg²⁺ cations for cleavage chemistry (9), typical of RNase H fold-mediated enzymes (24, 25). Structural studies have also been extended to binary complexes of both human (5, 6) and yeast (7) Agos bound to 5′-phosphorylated guide RNA strands.

Despite these singular advances in the structural biology of RNA silencing, further progress was hampered by the modest resolution (2.8- to 3.0-Å resolution) of TtAgo ternary complexes with guide DNA (4) and added target RNAs (8, 9). This precluded identification of water molecules coordinated with the pair of Mg²⁺ cations, including the key water that acts as a nucleophile and targets the cleavable phosphate between positions 10′-11′ on the target strand. We have now extended our research to TtAgo ternary complexes with guide DNA and target DNA strands, which has permitted us to grow crystals of ternary complexes that diffract to higher (2.2–2.3 Å) resolution in the cleavage-incompatible, cleavage-compatible, and postcleavage steps. These high-resolution structures of TtAgo ternary complexes provide snapshots of distinct key steps in the catalytic cleavage pathway, opening opportunities for experimental probing into DNA target cleavage as a defense mechanism against plasmids and possibly other mobile elements (26, 27).

Results

Ago Ternary Complexes with Target DNA in Cleavage-Incompatible State.

This section outlines the structures of Ago ternary complexes with 5′-phosphorylated 21-mer guide DNA and target DNAs (12 and 15 mers in length) that adopt a cleavage-incompatible state. We have solved the 2.8-Å crystal structure of the ternary complex of TtAgo bound to 12-mer target DNA (schematic in Fig. 1A; structure in Fig. 1B; X-ray statistics in SI Appendix, Table S1). The guide DNA strand (in red), which can be traced from positions 1–12 and 20–21, is anchored at both its ends, with the 5′-end anchored in the Mid pocket (14, 28) and the 3′-end in the PAZ pocket (29, 30). The target DNA strand (in blue) can be traced from positions 1′ to 12′, with guide-target pairing spanning base-pair positions 2–2′ to 12–12′, thereby encompassing both the seed region (base pairs 2–2′ to 8–8′) and the cleavage site (10′–11′ step on the target strand). Terminal base pair 2–2′ of the guide-target duplex is stacked over the side chains of Arg446 and His445 with bases 1 of the guide and 1′ of the target splayed out relative to the 2–2′ base pair and positioned in separate pockets in the Mid and PIWI domains, respectively (Fig. 1C). The positioning of the 5′-phosphate (14, 28) and sequence-specific recognition of T1 (9, 31, 32) of the guide strand in the Mid pocket is shown in Fig. 1D and SI Appendix, Fig. S1A. The sequence-specific recognition of G1′ on the target strand within its PIWI domain pocket is shown in Fig. 1E and SI Appendix, Fig. S1B.

Fig. 1.

Crystal structure and interactions in the TtAgo ternary complex with 5′-phosphorylated 21-mer guide DNA and 12-mer target DNA complementary to segment 2–12 of the guide strand in Mg²⁺-containing solution. (A) The sequence and pairing of guide (red) and target (blue) strands in the ternary complex. Disordered segments are shown in gray. (B) A 2.9-Å crystal structure of the complex. The various domains and linkers of TtAgo are color-coded, as are the guide and target strands. The catalytic residues in a stick representation are highlighted in a red-dotted background. (C) A view of the guide-target segment highlighting splaying out of 1 and 1′ bases in their respective Mid and PIWI pockets in the complex. The 2–2′ base pair stacks over side chains of Arg446 and His445. (D) Positioning of the 5′-phosphate and sequence-specific recognition of splayed-out base T1 of the guide strand in the Mid pocket. (E) Positioning and sequence-specific recognition of the splayed-out base G1′ of the target strand within a pocket in the PIWI domain. (F) Positioning of Glu512 outside and far away from the catalytic pocket composed of Asp478, Asp546, and Asp660 residues in the ternary complex.

The recent structural studies on the binary complex of yeast Ago with a bound 5′-phosphorylated guide RNA strand established that the catalytic pocket is made up of three Asp residues, with an inserted Glu completing a catalytic tetrad reflective of formation of a cleavage-compatible state (7). In the current structure of the TtAgo ternary complex with 12-mer target DNA, highly conserved Glu512 is positioned outside and far from the catalytic pocket made up of Asp478, Asp546, and Asp660 (Fig. 1 B and F), indicative of the formation of a cleavage-incompatible conformation at the 12-mer target DNA level.

We next compared the previously determined TtAgo ternary complex with 12-mer target RNA (8) and the current TtAgo ternary complex with 12-mer target DNA. The Ago-bound DNA–RNA (in gold) and DNA–DNA (in blue) duplexes superpose quite well (SI Appendix, Fig. S1C) with the DNA–DNA duplex (and DNA–RNA duplex), adopting a helical conformation closer to the canonical A-form (SI Appendix, Fig. S2A) than to the B-form (SI Appendix, Fig. S2B). Similarly, the Ago proteins in the two complexes (same color code) also superpose reasonably well (SI Appendix, Fig. S1D), and both adopt the cleavage-incompatible conformation, with small differences restricted to the positions of the PAZ domain (indicated by a red arrow, SI Appendix, Fig. S1D).

Ago Ternary Complex with Intact Target DNA in a Cleavage-Compatible State.

This section outlines the structures of Ago ternary complexes with 5′-phosphorylated 21-mer guide DNA and target DNAs (16 and 19 mers in length) that adopt a cleavage-competent state. We have solved the 2.2-Å crystal structure of the ternary complex of TtAgo bound to a 19-mer target DNA (schematic in Fig. 2A; X-ray statistics in SI Appendix, Table S2). There are two molecules of the complex in the asymmetric unit with the target DNA cleaved (between positions 10′ and 11′ with retention of the duplex on both sides of the cleavage site) in one molecule, whereas the density in the other molecule could be fit only on invoking a mixture of intact and cleaved target DNA at the 10′–11′ step (data analysis and electron density maps outlined and discussed in SI Appendix).

Fig. 2.

Crystal structure and interactions in the TtAgo ternary complex with 5′-phosphorylated 21-mer guide DNA and 19-mer target DNA complementary to segments 2–19 of the guide strand in Mg²⁺-containing solution. (A) The sequence and pairing of guide (red) and target (blue) strands in the ternary complex. (B) A 2.2-Å crystal structure of the complex. (C) Insertion of Glu512 into the catalytic pocket composed of Asp478, Asp546, and Asp660 residues in the ternary complex. (D–F) Conformational changes in loop L1 (D), in loop L2 that contains Glu512 (E), and in loop L3 that contains Asp546 (F) on proceeding from the cleavage-incompatible ternary complex with 12-mer target DNA to the cleavage-compatible ternary complex with 19-mer target DNA. (G and H) Relative positioning of loops L1 (in gold), L2 (in magenta), and L3 (in cyan) in a surface representation on proceeding from the cleavage-incompatible ternary complex with 12-mer target DNA (G) to the cleavage-compatible ternary complex with 19-mer target DNA (H).

In this section, we focus on the structure of the ternary complex containing an intact 19-mer DNA target (Fig. 2B) and discuss the structure of the ternary complex containing the cleaved DNA target in the next section. The guide DNA strand (in red), which can be traced from positions 1–16, is anchored at its 5′-end in the Mid pocket, but the 3′-end (residues 17–21 are disordered) is positioned too far from the PAZ pocket for insertion. The target DNA strand (in blue) can be traced from positions 1′ to 16′, with guide-target pairing spanning 15 bp from base-pair position 2–2′ to 16–16′ within the nucleic acid-binding channel, with this duplex also adopting a helical conformation closer to the canonical A-form (SI Appendix, Fig. S2E) than the B-form (SI Appendix, Fig. S2F).

Again, we compared the previously determined TtAgo ternary complex with 19-mer target RNA (9) and the current TtAgo ternary complex with 19-mer target DNA. The DNA–RNA and DNA–DNA duplex segments superpose reasonably well (SI Appendix, Fig. S3A), as do the Ago proteins in the two complexes, except for small differences in the PAZ domain (SI Appendix, Fig. S3B).

An important difference in the TtAgo ternary complex with 19-mer target DNA (relative to the complex with 12-mer target DNA) is the transition of Glu512 over a distance of 12.8 Å into the catalytic pocket to form a tetrad with the other three catalytic Asp residues (Fig. 2 B and C), thereby representing a cleavage-compatible conformation. We observed conformational changes on proceeding from the cleavage-incompatible ternary complex with 12-mer target DNA to the cleavage-compatible complex with 19-mer DNA (see segment in red box in SI Appendix, Fig. S4). Specifically, large structural transitions are observed within all three loops (Fig. 2 D–F; loops in ternary complexes with 12- and 19-mer target DNAs are colored in silver and gold, respectively), with Glu512 positioned on loop L2 inserting into the catalytic pocket (Fig. 2E).

The repositioning of the three loops relative to the 10′–11′ cleavage site on the target strand is clearly visible in the surface representations of the three loops (L1 in gold, L2 in magenta, and L3 in cyan) on proceeding from the cleavage-incompatible (Fig. 2G, ternary complex with 12-mer target DNA) to the cleavage-compatible (Fig. 2H, ternary complex with 19-mer target DNA) states. Moreover, additional protein–DNA intermolecular interactions are also observed with both the guide and target strands on proceeding from the cleavage-incompatible state (shown schematically for complexes with 12-mer target DNA in SI Appendix, Fig. S5A and with 15-mer target DNA in SI Appendix, Fig. S5B) to the cleavage-compatible state (complex with 19-mer target DNA, Fig. 3A).

Fig. 3.

Structural insights from studies of TtAgo ternary complexes with 5′-phosphorylated 21-mer guide DNA and added 19-mer target DNA in the presence of Mg²⁺ and Mn²⁺-containing solution. (A) Intermolecular contacts in the 2.2-Å ternary complex with cleavage-compatible 19-mer target DNA in Mg²⁺-containing solution. The interactions highlighted by a yellow background are additional contacts observed beyond those observed in the ternary complex with cleavage-incompatible 15-mer target DNA (SI Appendix, Fig. S5B). (B) Stereoview of the catalytic pocket in the ternary complex with an intact 10′–11′ step on the target strand in Mg²⁺-containing solution. The pair of Mg²⁺ cations are labeled “A” and “B” and are shown as magenta balls. Water molecules are shown as pink balls. The four catalytic Asp478, Asp546, Asp660, and Glu512 are shown in stick representation. (C) Stereoview of the catalytic pocket in the ternary complex with a cleaved 10′–11′ step on the target strand in Mg²⁺-containing solution. (D) A 2.4-Å crystal structure and interactions in the TtAgo ternary complex with 5′-phosphorylated 21-mer guide DNA and cleaved 19-mer target DNA complementary to segment 2–19 of the guide strand for crystals grown in Mn²⁺ containing solution. The target DNA is cleaved at the 10′–11′ step on the DNA target strand. The Inset expands the catalytic pocket segment showing the cleavage of the backbone.

In essence, release of the 3′-end of the guide strand from the PAZ pocket during the propagation step is accompanied by conformational transitions in loops 1, 2, and 3 with loop 2 residues, especially E512, forming stabilizing interactions on formation of the “inside” conformation. Such stabilization provides the driving force to shift E512 from the “outside” to the inside conformation, thereby positioning it in the catalytic pocket to complete tetrad formation.

We observe two hydrated Mg²⁺ cations (labeled A and B) that bridge between the three catalytic Asp residues and the cleavable but intact phosphate at the 10′–11′ step on the target strand (stereoview in Fig. 3B) in the cleavage-compatible state of the ternary complex with 19-mer target DNA. Mg²⁺ cation A is directly coordinated with Asp478 and Asp660 to a nonbridging phosphate oxygen and three water molecules, one of which (see arrow) is poised for in-line attack on the backbone phosphate at the cleavage site (Fig. 3B). Mg²⁺ cation B is directly coordinated with Asp478 and Asp546, with one each of bridging and nonbridging phosphate oxygens at the cleavage site, and with two water molecules (Fig. 3B). Notably, the carboxylate oxygens of Glu512, the inserted catalytic residue, are not directly coordinated to either divalent cation, but rather use a pair of bridging water molecules to coordinate with Mg²⁺ cation B (Fig. 3B). Given the 2.2-Å resolution of this complex, we can readily trace the Mg²⁺-coordinated water molecules, including the proposed nucleophilic water molecule within omit maps showing the positions of water molecules (SI Appendix). The coordination geometries around Mg²⁺ cations A and B for the intact ternary complex are shown in SI Appendix, Fig. S6A.

Ago Ternary Complex with Cleaved Target DNA.

The ternary complex of TtAgo bound to 5′-phosphorylated 21-mer guide DNA and cleaved 19-mer target DNA (between positions 10′ and 11′) is shown in Fig. 3D, with the cleavage-site segment magnified in the Inset. Importantly, we observed intact guide-target duplex segments on either side of the cleaved target DNA strand.

The divalent metal coordination geometry in the TtAgo ternary complex with cleaved 19-mer target DNA at the 10′–11′ step is shown in a stereoview in Fig. 3C. Mg²⁺ cation A is coordinated with Asp478 and Asp660 to a pair of nonbridging phosphate oxygens of the newly generated 5′-phosphate and two water molecules. Mg²⁺ cation B is coordinated with Asp478 and Asp546 to one nonbridging oxygen of the newly generated 5′-phosphate and the oxygen of the newly generated 3′-OH group and two water molecules. The inserted Glu512 remains coordinated with Mg²⁺ cation B through two bridging water molecules (Fig. 3C). The coordination geometries around Mg²⁺ cations A and B for the cleaved ternary complex are shown in SI Appendix, Fig. S6B.

We have superposed the structures encompassing the catalytic pocket of TtAgo ternary complexes containing intact (in gold) and cleaved (blue) DNA target sites with the superposition shown in stereo in SI Appendix, Fig. S7.

We also grew crystals of the TtAgo ternary complex with added 19-mer target DNA in Mn²⁺-containing solution under two different conditions. Our choice of Mn²⁺ reflected the preference for Mn²⁺ over Mg²⁺ as a divalent cation for cleavage (15). The 2.4-Å crystals of the ternary complex grown in Mn²⁺-containing solution yielded a structure (X-ray statistics in SI Appendix, Table S3) similar to that reported above for the cleaved structure of the ternary complex in Mg²⁺-containing solution (Fig. 3D). However, when the ternary complex in the Mn²⁺-containing solution was incubated at 55 °C for 10 min before setting up crystallization trays, not only did the 3.0-Å crystal structure of this complex (X-ray statistics in SI Appendix, Table S3) show cleavage of the target DNA strand at the 10′–11′ step, but also duplex formation was retained only on the side containing the seed segment, with the guide strand also disordered beyond position 10.

Comparison of Ago Ternary Complexes with 15-mer Target DNA and RNA.

The 2.25-Å crystal structure of the ternary complex of TtAgo bound to 5′-phosphorylated 21-mer guide DNA and 15-mer target DNA (schematic in Fig. 4A; X-ray statistics in SI Appendix, Table S1) is shown in Fig. 4B. The guide DNA strand (in red), which can be traced from positions 1–14 and 20–21, is anchored at both its ends. The target DNA strand (in blue) can be traced from positions 1′ to 14′, with guide-target pairing spanning 13 bp between base-pair positions 2–2′ to 14–14′.

Fig. 4.

Crystal structure and interactions in the TtAgo ternary complex with 5′-phosphorylated 21-mer guide DNA and 15-mer target DNA complementary to segments 2–15 of the guide strand in Mg²⁺-containing solution. (A) The sequence and pairing of guide (red) and target (blue) strands in the ternary complex. (B) A 2.25-Å crystal structure of the complex. (C) Positioning of Glu512 outside and far away from the catalytic pocket in the ternary complex with 15-mer target DNA. (D) Insertion of Glu512 into the catalytic pocket in the ternary complex with 15-mer target RNA reported previously (Protein Data Bank ID code 3HJF). (E) Superposition of the guide DNA-target DNA (in silver) and guide DNA-target RNA (in magneta) in the TtAgo ternary complexes containing 15-mer target DNAs and RNAs. Note that we observed one more base pair (15–15′) in the ternary complex with target RNA. (F) Superposition of the catalytic pockets and loops L1, L2, and L3 in the ternary complexes with target DNA (in silver) and target RNA (in magenta).

Importantly, in the TtAgo ternary complex with 15-mer target DNA (formation of 13 bp), where the 3′-end of the guide strand is retained within the PAZ pocket, Glu512 is positioned outside and far from the catalytic pocket (Fig. 4C), indicative of a cleavage-incompatible conformation. By contrast, in the TtAgo ternary complex with 15-mer target RNA (formation of 14 bp), where the 3′-end of the guide strand is released from the PAZ pocket (9), Glu512 is inserted into the catalytic pocket (Fig. 4D), indicative of a cleavage-compatible conformation. Thus, it appears that a 15-mer target strand represents the tipping point during the propagation step between cleavage-incompatible and cleavage-compatible conformations, with DNA targets of this length being cleavage-incompatible and their RNA counterparts adopting cleavage-compatible conformations. We are unclear as to the origin of this difference in behavior between 15-mer DNA and RNA targets other than to point out that the Ago ternary complex with 15-mer target DNA results in formation of a 13-bp guide DNA-target DNA duplex, and the Ago complex with 15-mer target RNA results in formation of a 14-bp guide DNA-target RNA duplex (Fig. 4E), both of which adopt A-like conformations.

The TtAgo ternary complexes formed with 15-mer target DNA (in silver) and 15-mer target RNA (in magenta) are superposed in SI Appendix, Fig. S8, with the observed conformational changes in the PAZ and N-domains and the catalytic pocket reflective of transitions associated with conversion from cleavage-incompatible (15-mer target DNA; 13-bp formation in silver) to cleavage-compatible (15-mer target RNA; 14-bp formation in magenta) conformations (duplexes superposed in Fig. 4E). The conformational changes of loops L1, L2, and L3 within the catalytic pocket associated with this transition are shown in Fig. 4F.

We also solved the 2.5-Å structure of TtAgo bound to 5′-phosphorylated guide DNA and 16-mer target DNA (X-ray statistics in SI Appendix, Table S2). This ternary complex formed a cleavage-compatible conformation involving formation of 15 bp of guide-target duplex similar to the ternary complex observed with the 19-mer target DNA. Thus, for TtAgo ternary complexes with DNA target strands, the switch from cleavage-incompatible to cleavage-compatible complexes occurs on transition from 15-mer (formation of 13 bp) to 16-mer (formation of 15 bp) target DNA pairing with the guide DNA during the propagation step.

Discussion

Cleavage Mechanism.

The PIWI domain of Ago/PIWI proteins has been shown to adopt an RNase H fold as first established from structural studies of Pyrococcus furiosus Ago (10) and Archaeoglobus fulgidus PIWI (11) in the free state. The catalytic mechanism of RNase H folds has been investigated in detail (reviewed in ref. 24) with the emphasis on factors that contribute to substrate specificity and the key contribution of a pair of Mg²⁺ cations to cleavage chemistry (33). The pair of Mg²⁺ cations, which are positioned to bridge the nucleic acid substrate and enzyme catalytic residues when properly aligned, have been proposed to greatly enhance both substrate recognition and product release, thereby enhancing catalytic efficiency. Furthermore, the scissile phosphate group projects three coordination ligands to the pair of Mg²⁺ cations in the enzyme–substrate complex. Of the pair of Mg²⁺ cations in the catalytic pocket, cation A assists in nucleophilic attack by activating a water molecule for in-line attack on the cleavable phosphate, whereas cation B stabilizes the pentacovalent intermediate and facilitates the protonation of the 3′ oxyanion-leaving group by a water molecule (reviewed in ref. 24).

Previous studies of TtAgo complexes with 5′-phosphorylated guide DNA and complementary target RNAs yielded structures of ternary complexes that diffracted at best to a 2.6-Å resolution. Thus, although we were able to identify the position of Mg²⁺ cations, we were unable to identify the water molecules coordinated with these Mg²⁺ cations and hence were limited in our efforts at deducing insights into the mechanism of cleavage chemistry (9). Nevertheless, inspired by previously reported gel-based cleavage studies of TtAgo ternary complexes using guide and target DNAs (9), we have attempted to improve the diffraction quality of our TtAgo ternary complexes by switching from target RNAs to their target DNA counterparts. This approach has yielded higher-resolution crystals of ternary complexes that diffract to 2.25 Å (with 15-mer target DNA; Fig. 4B) and 2.2 Å (with 19-mer target DNA; Fig. 2B), thereby allowing us to identify Mg²⁺-coordinated water molecules, including the one coordinated with Mg²⁺ cation A and positioned for in-line nucleophilic attack on the cleavable phosphate (Fig. 3B). Furthermore, the set of structures of ternary complexes solved in this study provide snapshots of cleavage-incompatible, cleavage-compatible, and postcleavage states, as well as the conformational transitions required for formation of a cleavage-compatible pocket.

Features of the catalytic cleavage mechanism of Ago ternary complexes with guide and target DNAs are summarized in Fig. 5. In the cleavage-incompatible conformation of the ternary complex with 12- and 15-mer target DNAs, the three catalytic Asp residues at positions 478, 546, and 660 are not properly positioned relative to the cleavable phosphate, with Glu512 positioned outside and far from the catalytic pocket and an absence of a pair of Mg²⁺ cations (Fig. 5A).

Fig. 5.

A proposed mechanism for Ago-mediated Mg²⁺ cation-dependent cleavage of target strands. Crystal structure snapshots (A, B, and D) and a proposed model of transition state (C) in the reaction pathway leading to cleavage of the target DNA strand at the 10′–11′ step in the ternary complex of TtAgo with complementary guide and target DNA strands. (A) Structure of the catalytic pocket in the cleavage-incompatible ternary complex with Glu512 positioned outside and far from the catalytic pocket as observed in ternary complexes with 12- and 15-mer target DNA strands. (B) Structure of the catalytic pocket in the cleavage-compatible ternary complex with Gln512 inserted into the catalytic pocket as observed in the ternary complex with 16- and 19-mer target DNA strands. (C) Proposed model of the transition state of the cleavage reaction in the ternary complex. (D) Structure of the catalytic pocket of the ternary complex following cleavage of the 10′–11′ backbone in the ternary complex with cleaved 19-mer target DNA strand.

We highlight the following conformational changes on proceeding from the cleavage-incompatible (target DNAs of length 12–15 mer) to cleavage-compatible (target DNAs of length 16–19 mer) states. Thus, the transition from ternary complex with 15-mer target DNA (13 bp of guide-target duplex) to ternary complex with 16-mer target DNA (15 bp of guide-target duplex) during the propagation step results in the release of the 3′-end of the guide from the PAZ pocket. The accompanying conformational changes both in the guide strand and in loops L1, L2, and L3 positions Glu512 for insertion into the catalytic pocket, in the process forming a cleavage-compatible precleavage state with components held in optimal position for cleavage by two bound Mg²⁺ cations (Fig. 5B). The coordination geometries of Mg²⁺ cations A and B with Asp side chains, the phosphate oxygens, and coordinated waters, including the nucleophilic water poised for attack on the cleavable phosphate (shown by a red arrow Fig. 5B), in the precleavage structure of the complex are those characteristic of RNase H enzymes (reviewed in ref. 24). The pair of Mg²⁺ cations are separated by 3.7 Å with octahedral-like coordination geometries for Mg²⁺ cations A and B, as shown in SI Appendix, Fig. S6A. One difference is that Glu512 is coordinated with Mg²⁺ cation B through a pair of bridging water molecules in our Ago ternary complexes (Fig. 5B), in contrast with direct coordination with Mg²⁺ cation B in canonical RNase H enzymes (reviewed in ref. 24). Importantly, the nucleophilic water, the cleavable phosphate, and the O3 phosphate-leaving group are positioned for in-line attack to generate the proposed pentacovalent phosphate transition state associated with an SN2 reaction with inversion of stereochemistry at the cleavable phosphate position (model of transition state shown in Fig. 5C).

The structure of the postcleavage state of the ternary complex is shown in Fig. 5D. Here again, the coordination geometries of Mg²⁺ cations A and B with Asp side chains, the phosphate oxygens, and coordinated waters are those characteristic of RNase H enzymes in the postcleavage state (reviewed in ref. 24). The pair of Mg²⁺ cations are separated by 3.6 Å, such that Mg²⁺ cation B moves toward A, with octahedral-like coordination geometries for Mg²⁺ cations A and B, as shown in SI Appendix, Fig. S6B. In our postcleavage structures of the ternary complex, Glu512 remains coordinated through two bridging water molecules to Mg²⁺ cation B.

DNA Guide Strand-Mediated DNA Target Cleavage.

Our previous in vitro biochemical (9) and the current structural studies highlight the ability of bacterial Agos to use guide DNA strands to cleave target DNA strands. Such results suggest the existence of a bacterial Argonaute-mediated DNA interference pathway as a defense mechanism against transposons and mobile genetic elements, first proposed from bioinformatic studies of Ago proteins (34) and recently receiving support from molecular biology experiments (26, 27).

Materials and Methods

TtAgo was prepared and purified as described previously (4) with ternary complexes generated by adding 5′-phosphorylated 21-mer guide DNA and target DNAs of varying length in the presence of divalent cations. All data sets were integrated and scaled with the HKL2000 suite (35). All structures were solved by molecular replacement with the program PHASER (36), and refined by COOT (37) and PHENIX (38). Details of crystallization, data collection, and structure determination and refinement are provided in SI Appendix. The refinement statistics for the ternary complexes are summarized in SI Appendix, Tables S1–S3.