Structural features of Argonaute-GW182 protein interactions

Abstract

MicroRNAs (miRNAs) guide Argonaute (Ago) proteins to target mRNAs, leading to gene silencing. However, Ago proteins are not the actual mediators of gene silencing but interact with a member of the GW182 protein family (also known as GW proteins), which coordinates all downstream steps in gene silencing. GW proteins contain an N-terminal Ago-binding domain that is characterized by multiple GW repeats and a C-terminal silencing domain with several globular domains. Within the Ago-binding domain, Trp residues mediate the direct interaction with the Ago protein. Here, we have characterized the interaction of Ago proteins with GW proteins in molecular detail. Using biochemical and NMR experiments, we show that only a subset of Trp residues engage in Ago interactions. The Trp residues are located in intrinsically disordered regions, where flanking residues mediate additional weak interactions, that might explain the importance of specific tryptophans. Using cross-linking followed by mass spectrometry, we map the GW protein interactions with Ago2, which allows for structural modeling of Ago-GW182 interaction. Our data further indicate that the Ago-GW protein interaction might be a two-step process involving the sequential binding of two tryptophans separated by a spacer with a minimal length of 10 aa.

Keywords: RNA interference; RNAi; gene regulation; small RNA–mediated gene silencing.

Fig. 1.

Identification of Ago2 binding sites on TNRC6B. (A) Domain organization of Ago2 and Coomassie-stained recombinant Ago2. (B) Schematic illustration of the peptide array used in C. (C) Domain organization of TNRC6B. Parts of the sequence, indicated as A-I, were spotted on the membrane. TNRC6B peptides were blotted onto a nitrocellulose membrane and Ago2 binding was assessed as shown in B. Signals were quantified using the ImageJ software (http://rsb.info.nih.gov/ij), showing different levels of affinity (right image). (D) List of TNRC6B peptides with affinity for Ago2. The strongest Ago2-binding peptides are marked with filled circles. (E) Residues directly flanking Trp found in the individual peptides.

Fig. 2.

Analysis of residues neighboring tryptophans. (A) Peptide array showing different effects of amino acid substitutions in peptide E11 (Lower). (Upper) The control experiment lacking Ago2 incubation. (B–F) List of designed peptides with the type of mutation pointed out in gray. Tryptophans are highlighted in blue. (B) Alanine scan. (C) Systematic introduction of multiple alanines. (D) A single amino acid in a poly Gly peptide and change of GWG position in peptide E11. (E) Trp substitution by aromatic (F, Y) or selected other (L, R) amino acids and peptides used for STD-NMR measurements in Fig. 3. (F) Change of W neighboring amino acids.

Fig. 3.

STD-NMR identifies Trp protons engaged in Ago2 binding. (A) Schematic illustration of STD-NMR experiment applied to the protein receptor Ago2 and ligand (peptide) complex. Selective saturation of receptor signals is transferred to the peptide protons by spin diffusion. The stronger the receptor–ligand contact between two protons, the stronger is the STD effect. (B) STD amplification factor of measured peptide (defined in Materials and Methods). (Left) Time course of STD amplification factors plotted against the saturation transfer time calculated for peptide E11. Different residues are highlighted by colors, whereas different symbols are used for protons observed. (Right) Structure of tryptophans with proton names annotated. The value of the proton with the highest STD amplification factor was set to 100% and is highlighted in red and relative values are then indicated for the other protons, accordingly. (C) Trp-binding pocket of W901 (PDB ID code 4EI3).

Fig. 4.

Folding analysis of a TNRC6B peptide that binds Ago2. (A) TNRC6B orthologs were aligned with MAFFT (43) using the Jalview software (44). Conserved sequence motifs are shaded in blue. Predicted secondary structure elements are displayed above the sequence. (B) Coomassie-stained SDS/PAGE of recombinant TNRC6B-599-683 used in the subsequent experiments. (C) CD spectrum of TNRC6B-599-683 reveals a random coil-like structure. (D) Heteronuclear NOE experiments of TNRC6B-599-683. The average value for heteronuclear NOE is about 0.4, as indicated with a gray line. For secondary structural elements, a value above 0.77 would be expected, as marked with a dashed line. Secondary structure analysis based on the ¹³C secondary chemical shifts of the free TNRC6B-599-683 fragment indicating the absence of significant population of secondary structure.

Fig. 5.

(A) Pull down of proteins from HeLa lysate by TNRC6B-599-683. Whole-cell HeLa lysate was incubated with GST-tagged TNRC6B-599-683, followed by a GST pull down. The ratios of precipitated Ago fractions were determined by mass spectrometry. (B) DLS indicates that TNRC6B-599-683 and Ago2 do not aggregate upon binding. DLS data show small particles for TNRC6B-599-683 and Ago2 alone (R_H: 1.34 and 3.05 nm, respectively), as well as for the complex (R_H: 1.17 and 3.78 nm). Intensities were normalized for molecular weight as indicated by a red mark. (C) FP measurement of TNRC6B-599-683:Ago2 interaction provides a K_D of 1.87 ± 0.47 µM (mean and deviation were calculated from a triple measurement for each sample).

Fig. 6.

(A) NMR titration experiments of TNRC6B-599-683 and Ago2. Amino acids colored in gray could not be assigned. The unambiguously assigned Trp623 and Gly624 are marked by an asterisk. (Upper) Plot of peak intensity ratios of a 0.1:1 (gray) and 1:1 (black) TNRC6B-599-683–Ago2 compared with the free TNRC6B-599-683 reference spectrum. Reduced intensity is observed for residues 606–634. (Lower) CSPs correspond to the region with reduced intensity ratios. (B) Ago2 interacts with two distinct tryptophans. (Upper) Input for pull-down assays with respective mutants indicated on top. (Lower) Ago2 precipitation by GST-pull-downs with GST–TNRC6B-599-683, as well as the indicated mutants, were immobilized and used for Ago2 pull-down experiments. Molecular-weight markers are shown to the left of the gels. (C) Mutation of W623 in wt TNRC6B reduces Ago2 binding. F/H-TNRC6B, -TNRC6B W623A, -TNRC6B W623/634A, and -GFP were transfected into HEK 293 cells, immunoprecipitated, and analyzed by Western blotting against endogenous Ago2 (Top) or the tagged proteins (Middle and Bottom).

Fig. 7.

Protein–protein cross-links identify the TNRC6B interaction surface on Ago2. (A) SDS/PAGE of cross-linked TNRC6B-599-683-Ago2 complex before (cross-link) and after separation by size-exclusion chromatography (GF peak). The purified sample was subjected to MS analysis. (B) After cross-linking of TNRC6B-599-681 and Ago2, the complex can be separated from free TNRC6B-599-683 (blue line) and is slightly shifted toward lower elution volume in comparison with free Ago2 (light blue line). (C) The cross-link map illustrates the cross-links found between Ago2 and TNRC6B-599-683 (violet) and the Ago2 intramolecular cross-links (black dashed lines). A detailed list of cross-linked peptides is shown in Fig. S4. (D) Surface presentation of the Ago2 crystal structure with cocrystallized Trp (cyan) (PDB ID code 4EI3) (28). Lys on Ago2 that cross-linked with TNRC6B Lys (K425, K655, K693, K844) is highlighted in dark red. All other lysines present in the Ago2 sequence are presented in pink to show possible cross-linking sites. (E) Spacer variations used in F. (F) GST-tagged spacer variants indicated in E were incubated with recombinant Ago2. Protein complexes were isolated by GST pull down and analyzed by SDS/PAGE, followed by Coomassie staining. (Upper) Input samples. (Lower) Pulled-down proteins.

Fig. 8.

Schematic model of the Ago2–TNRC6B-599-683 binding mechanism. In a first step, TNRC6B-599-683 binds with its first Trp (W623) to one Ago2 Trp-binding pocket. All lysines in the TNRC6B-599-683 sequence remain flexible and can reach Ago2 Lys for cross-linking. In a second step, binding of the second Trp (W634) to the other pocket completes the binding and increases affinity. The two-step mechanism explains all cross-links that were observed (Ago2 Lys are indicated in red and cross-links as dotted lines).