The evolutionary journey of Argonaute proteins

Abstract

Argonaute proteins are conserved throughout all domains of life. Recently characterized prokaryotic Argonaute proteins (pAgos) participate in host defense by DNA interference, whereas eukaryotic Argonaute proteins (eAgos) control a wide range of processes by RNA interference. Here we review molecular mechanisms of guide and target binding by Argonaute proteins, and describe how the conformational changes induced by target binding lead to target cleavage. On the basis of structural comparisons and phylogenetic analyses of pAgos and eAgos, we reconstruct the evolutionary journey of the Argonaute proteins through the three domains of life and discuss how different structural features of pAgos and eAgos relate to their distinct physiological roles.

Figure 1

Domain architectures of the PIWI superfamily proteins. Dotted lines indicate separate genes located in the same (predicted) operon. *, Ago proteins with an incomplete DEDX catalytic tetrad in the PIWI domain. Guide and target usage is based on available biochemical data (underlined) or predicted (in parentheses). **, predicted nucleases from Sir2, Mrr or TIR protein families. ***, predicted nucleases from Sir2, Mrr, Cas4 or PLD protein families. REase, restriction endonuclease; DExD/H, superfamily II helicase (denoted after a signature amino acid motif).

Figure 2

TtAgo with 21-mer guide DNA (binary complex) and with complementary 12-mer target RNA (ternary complex) adopt cleavage-incompatible conformations. (a) Domain architecture of TtAgo. (b) Sequence of 5′-phosphorylated guide DNA (red, with disordered segment in gray and seed segment underlined) and complementary 12-mer target RNA (blue). (c–g) 3.0 Å structure of the binary complex of TtAgo bound to 5′-phosphorylated 21-mer guide DNA (PDB 3DLH). TtAgo in ribbon representation, domains colored as in a; guide DNA in red, in stick representation. (c) Overall view. (d) Insertion of the 5′-phosphate of the guide DNA into the MID pocket. (e) Insertion of the 2-nt 3′-end of the guide DNA into the PAZ pocket. (f) Outward directionality of bases 2–6 of the guide DNA in the binary complex of TtAgo with guide DNA, thereby aligning their Watson-Crick edges for pairing with target nucleic acids. (g) Bases 10 and 11 of the guide DNA are splayed apart as a result of insertion of an arginine side chain. (h,i) 2.6 Å structure of the ternary complex of TtAgo(D546N) bound to 5′-phosphorylated 21-mer guide DNA and complementary 12-mer target RNA (PDB 3HO1). Guide DNA (red) and target RNA (blue) are in stick representation. (h) Overall view. (i) Bases 10 and 11 of the guide DNA are stacked.

Figure 3

TtAgo with 5′-phosphorylated 21-mer guide DNA and complementary 15-mer and 19-mer target RNAs (ternary complex) adopt cleavage-compatible conformations. (a) Sequences of 5′-phosphorylated guide DNA (red, with disordered segment in gray and seed segment underlined) and complementary 15-mer target RNA (blue). (b) 3.0 Å ternary complex of TtAgo(D546E) bound to guide DNA and 15-mer target RNA (PDB 3HJF). The guide DNA and target RNA are in a stick representation, with same colors as in a. (c–e) Conformational changes in TtAgo during its transition from ternary complex with 12-mer target RNA in a cleavage-incompatible conformation (silver; PDB 3HO1) to ternary complex with 15-mer target RNA in a cleavage-compatible conformation (magenta; PDB 3HJF). (c) Rotation of the PAZ domain. (d) Transitions in loops PL1, PL2 and PL3. (e) Rearrangement of the β-strand (Gly489 to Val494) of TtAgo by one residue and conformational transition in adjacent loop PL1. (f) Sequences of 5′-phosphorylated guide DNA (red, with disordered segment in gray and seed segment underlined) and complementary 19-mer target RNA (blue). (g) 2.8 Å ternary complex of TtAgo (D478A mutant) bound to guide DNA and 19-mer target RNA (PDB 3HK2). (h) The N domain blocks base pairing of the guide and the 19-mer target RNA beyond position 16 of the target strand.

Figure 4

Structure-based insights into the cleavage mechanism of TtAgo. (a,b) Positioning of Glu512 (surface shown in a dotted representation) of TtAgo in the ternary complexes with 5′-phosphorylated 21-mer guide DNA and complementary 12-mer target DNA (a; Glu512 outside and directed away from the catalytic pocket, representative of a cleavage-incompatible conformation; PDB 4N47) and 19-mer target DNA (b; Glu512 inserted into the catalytic pocket, representative of a cleavage-compatible conformation; PDB 4NCB). (c–f), Proposed mechanism for Ago-mediated Mg²⁺-coordinated cleavage of target strand at the 10′–11′ step in the ternary complex of TtAgo with complementary DNA guide and DNA target strands. Crystal structure snapshots show cleavage-incompatible (c; PDB 4N47), cleavage-compatible (d; PDB 4NCB) and post-cleavage (f; PDB 4N76) states, along with with a proposed model of the transition state (e).

Figure 5

Structures of binary complexes of KpAgo and hAGO2 bound to 5′-phosphorylated guide RNAs. (a) 3.2 Å structure of KpAgo (ribbon representation) with fortuitously loaded 5′-phosphorylated guide RNA (red, stick representation; PDB 4F1N). (b) 2.3 Å structure of hAGO2 (ribbon representation) with fortuitously loaded 5′-phosphorylated guide RNA (red, stick representation; PDB 4EI1). (c–e) Details of the 2.2 Å structure of hAGO2 bound to a defined, miR-20a 5′-phosphorylated guide RNA (PDB 4F3T). (c) Insertion of Ile365 (dotted circle), projecting from α-helix 7 of hAGO2, between bases 6 and 7 of the RNA guide strand. (d) Splaying apart of bases 9 and 10 of the guide RNA by insertion of Arg710 side chain. (e) Intermolecular contacts between 2′-OH groups of guide RNA and amino acid backbone and side chains of hAGO2; both direct and water-mediated (pink spheres) intermolecular hydrogen bonds are shown. (f,g) Intermolecular hydrogen bonding interactions stabilizing the conformation of the expanded and repositioned loop PL2 that inserts the glutamic acid finger into the catalytic pocket in the structure of the KpAgo binary complex with a fortuitously loaded 5′-phosphorylated guide RNA (f, PDB 4F1N) and in the structure of the TtAgo ternary complex with 5′-phosphorylated guide DNA and 19-mer target RNA (g, PDB 3HVR). (h) A pair of tryptophan-binding pockets on the surface of hAGO2 in its binary complex with a fortuitously loaded 5′-phosphorylated guide RNA (PDB 4EI3).

Figure 6

Phylogenetic trees of Argonaute proteins. (a,b) Maximum-likelihood phylogenetic unrooted trees were built using the FastTree program using a multiple alignment of conserved blocks of MID and PIWI domains. The same program was also used to compute the bootstrap values (percentages) that are indicated for all internal branches. Green, Bacteria; orange, Archaea; purple, Eukaryota. Collapsed branches are shown as triangles of the corresponding color. Organisms of which Agos are discussed in this manuscript are colored red. (a) Phylogenetic analysis of pAgos and organization of the predicted operons. We clustered 487 pAgo proteins identified in Refseq by sequence similarity and selected a nonredundant representative set (261 pAgos and 8 selected eAgos). Red arrows indicate two alternative roots of the pAgo tree. *, long pAgo clade contains several short pAgos. **, not all eukaryotic eAgos have an intact catalytic tetrad. Domains associated with pAgos are shown as boxes on the right side of the tree. Homologous domains are shown by boxes of the same color or pattern. Sir2 1 and Sir2 2 are two distinct families of the predicted Sir2-like nuclease; RE1 and RE2 are two distinct families of restriction endonuclease superfamily. TIR, predicted nuclease of TIP family; Schlafen, predicted ATPase; APAZ, ‘analog of PAZ’ domain; Cas4, Cas4 subfamily of restriction endonuclease superfamily; PLD, predicted nuclease of phospholipase D superfamily. Gray boxes indicate distinct families of uncharacterized proteins. Short and long pAgos are not shown but present in all the operons. Slashes denote ‘and’. pAgo sequence alignment and uncollapsed phylogenetic tree are in Supplementary Data 1 and 2, respectively, and are described in Supplementary Note. (b) Phylogenetic analysis of a representative set of 177 eAgos. 1, Trypanosoma Ago family; 2, WAGO family. eAgo sequence alignment and uncollapsed phylogenetic tree are in Supplementary Data 3 and 4, respectively, and are described in Supplementary Note.