Structure-based cleavage mechanism of Thermus thermophilus Argonaute DNA guide strand-mediated DNA target cleavage - PubMed (original) (raw)

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

Gang Sheng et al. Proc Natl Acad Sci U S A. 2014.

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 Mg(2+) 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.

Keywords: DNA guide-DNA target; bacterial Argonaute; catalytic mechanism.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Crystal structure and interactions in the _Tt_Ago 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 _Tt_Ago 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.

Fig. 2.

Fig. 2.

Crystal structure and interactions in the _Tt_Ago 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).

Fig. 3.

Fig. 3.

Structural insights from studies of _Tt_Ago 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. S5_B_

). (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 _Tt_Ago 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.

Fig. 4.

Fig. 4.

Crystal structure and interactions in the _Tt_Ago 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 _Tt_Ago 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).

Fig. 5.

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 _Tt_Ago 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.

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