Structure of the guide-strand-containing argonaute silencing complex

Abstract

The slicer activity of the RNA-induced silencing complex is associated with argonaute, the RNase H-like PIWI domain of which catalyses guide-strand-mediated sequence-specific cleavage of target messenger RNA. Here we report on the crystal structure of Thermus thermophilus argonaute bound to a 5'-phosphorylated 21-base DNA guide strand, thereby identifying the nucleic-acid-binding channel positioned between the PAZ- and PIWI-containing lobes, as well as the pivot-like conformational changes associated with complex formation. The bound guide strand is anchored at both of its ends, with the solvent-exposed Watson-Crick edges of stacked bases 2 to 6 positioned for nucleation with the mRNA target, whereas two critically positioned arginines lock bases 10 and 11 at the cleavage site into an unanticipated orthogonal alignment. Biochemical studies indicate that key amino acid residues at the active site and those lining the 5'-phosphate-binding pocket made up of the Mid domain are critical for cleavage activity, whereas alterations of residues lining the 2-nucleotide 3'-end-binding pocket made up of the PAZ domain show little effect.

Figure 1. Crystal structure of T. thermophilus argonaute bound to a 5′-phosphorylated 21-base DNA guide strand

a, Stick (DNA) and ribbon (Ago) view of the 3.0 Å structure of the complex. Individual domains and linkers are colour-coded. The bound 21-base DNA guide strand is coloured red (with phosphorus atoms in yellow), and can be traced for nucleotides 1 to 11 and 18 to 21, together with phosphates at positions 16 and 17. b, Corresponding stick (DNA) and electrostatically colour-coded surface (Ago) view of the complex. c, An alternate stereo view of the complex from that shown in a. For rotation of c, see Supplementary Movie 2DLH.

Figure 2. Intermolecular hydrogen-bonding alignments in the T. thermophilus Ago bound to a 21-base DNA guide strand

a, Insertion of the 5′-phosphate of the DNA guide strand into the binding pocket in the Mid domain. b, Positioning of stacked 3′-end residues 20 and 21 of the DNA guide strand into the binding pocket in the PAZ domain. c, Positioning of stacked residues 2 to 6 of the DNA guide strand, with emphasis on intermolecular interactions. d, Positioning of stacked residues 6 to 10 and 11 of the DNA guide strand, with emphasis on intermolecular interactions.

Figure 3. Conformational changes in T. thermophilus Ago on formation of the 21-base DNA guide strand complex

a, The 2.7 Å structure of T. thermophilus Ago bound to a 5′-phosphorylated 10-base DNA guide strand. The bound 10-base DNA can be traced for nucleotides 6 to 10. b, Superpositioning of the PAZ domains together with observable 3′ ends in DNA 21-base (in magenta) and 10-base (in green) guide strand complexes. c, Alignment of Agos in complexes with bound 10-base (in lighter shades) and 21-base (in darker shades) DNA guide strands, after superpositioning of their PAZ domains (boxed segment). The grey arrows indicate the magnitude of the conformational changes on proceeding from the 10-base to the 21-base complexes. For transition between a and Fig. 1c, see Supplementary Movie 3DLB–3DLH.

Figure 4. DNA-guide-dependent RNA cleavage activity of wild-type and mutant T. thermophilus Ago

A 5′-phosphorylated 21-nucleotide DNA oligonucleotide sequence was pre-incubated with the indicated Ago proteins followed by the addition of 5′-radiolabelled RNA cleavage substrate. Cleavage products were resolved using denaturing polyacrylamide gel electrophoresis gels and visualized by phosphoimaging. The black bar indicates the region of the cleavage substrate covered by the guide DNA, with the cleavage site shown by an arrow. a, Catalytic Asp mutants (left panel); b, Mid domain mutants (central left panel); c, PAZ domain mutants (central right panel); and d, the L1-catalytic domain interface mutants (right panel).