Multivalent Recruitment of Human Argonaute by GW182 Elad Elkayam, Christopher R. Faehnle, Marjorie Morales, Jingchuan Sun, Huilin Li, Leemor Joshua-Tor  Molecular Cell  Volume 67, Issue 4, Pages 646-658.e3 (August 2017) DOI: 10.1016/j.molcel.2017.07.007 Copyright © 2017 Elsevier Inc. Terms and Conditions

Molecular Cell 2017 67, 646-658.e3DOI: (10.1016/j.molcel.2017.07.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 1 hGW182 Argonaute-Binding Motifs Binding to hAgo2 and hAgo1 (A) Schematic domain organization of hGW182 (TNRC6A). The different Ago-binding motifs within the Ago-binding domain (ABD) are colored in yellow. (B and C) Isothermal titration calorimetry (ITC) analysis of the interaction between hGW182 Ago-binding motifs and hAgo2 (B) and hAgo1 (C). In all experiments, the ITC cell was filled with either hAgo2 or hAgo1 and the different hGW182 Ago-binding motifs were titrated as the ligands. Affinities are reported as dissociation constants (Kd) ± SEs calculated from the fit. (D and E) Fluorescence polarization (FP) binding experiments of FITC-labeled hGW182 hook motif with hAgo2 (D) and hAgo1 (E) showing increased affinity of the hook motif to both hAgo2 and hAgo1 that are loaded with endogenous RNA (solid blue line) compared to RNA-free hAgo2 and hAgo1 (dashed red line). The graphs show mean ± SD (n = 3). Dissociation constants (Kd) were calculated by fitting data from three different experiments and are shown as the average ± SD. See also Figure S1. Molecular Cell 2017 67, 646-658.e3DOI: (10.1016/j.molcel.2017.07.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 2 The Structure of the hGW182 Hook Motif in Complex with hAgo1-miRNA (A) Cartoon representation of the overall structure of the hAgo1-miRNA-hGW182 hook motif complex. The hGW182 hook (gold) is bound to the PIWI domain (purple) with the two tryptophans (in red) anchored in the hydrophobic GW-binding pockets (GWBPs). The N and C termini of the peptide are marked. (B) A close-up of hAgo GWBPs with the bound hGW182 hook motif. GWBP1 residues are colored in green and GWBP2 residues are colored in pink. The PIWI domain is colored in purple and the hook in gold. See also Figure S2 and Table 1. Molecular Cell 2017 67, 646-658.e3DOI: (10.1016/j.molcel.2017.07.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 3 The Open and Closed hGW182-Binding “Gate” (A) hAgo1 GWBP2 gate residues form a tight salt bridge upon binding of the hGW182 hook, thus closing the gate. (B) Open gate conformation in the absence of hGW182 hook binding as in the structure of hAgo1 with endogenous RNA (PDB: 4KRE). (C and D) Similar “gate” closure is observed in the presence of tryptophan in the structure hAgo2 in complex with tryptophan (PDB: 4OLB) (C) and in the presence of phenol in the hAgo2-miR20a complex (PDB: 4F3T) (D). See also Figure S3. Molecular Cell 2017 67, 646-658.e3DOI: (10.1016/j.molcel.2017.07.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 4 Mutational Analysis of the GW-Binding Pockets of hAgo1 and hAgo2 (A) Fluorescence polarization (FP) binding experiments of FITC-labeled hGW182 hook to hAgo2. (B) Same as (A) but with hAgo1. GW-binding-pocket mutants show drastic decrease in hGW182 hook binding when residues from both binding pockets are mutated simultaneously. Data shown are from three different experiments and presented as the average ± SD. The lines for fitted curves with Rsquare < 0.7 were omitted from the figure. (C) FP binding experiments of hAgo2 “gate” residue mutants with FITC-labeled hGW182 showing a substantial decrease in binding compared to wild-type. (D) Similar experiments for gate residue mutants of hAgo1. The graphs show mean ± SD (n = 3). Dissociation constants (Kd) were calculated by fitting data from three different experiments and are shown as average ± SD. See also Table S1. Molecular Cell 2017 67, 646-658.e3DOI: (10.1016/j.molcel.2017.07.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 5 All Three Argonaute-Binding Motifs of hGW182 Compete for the Same Binding Site on Both hAgo2 and hAgo1 (A) FP binding experiments of FITC-labeled hook motif binding to hAgo2 with increasing concentrations of motif-1 (red), motif-2 (green), and unlabeled hook (purple) of hGW182. (B) Same as (A) but for binding to hAgo1. The graphs show mean ± SD (n = 3). (C) Isothermal titration calorimetry (ITC) analysis of the interaction between the hGW182 hook motif and hAgo2 in the presence of 5 μM hGW182 motif-2. (D) Same experiment as in (C) but in the presence of 10 μM motif-2. The decrease in binding affinity of the hook motif to hAgo2 correlates with the increasing concentration of motif-2. Affinities are reported as dissociation constants (Kd) ± SEs calculated from the fit using a competitive binding model. Molecular Cell 2017 67, 646-658.e3DOI: (10.1016/j.molcel.2017.07.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 6 Tryptophan Residues in All Three Motifs of hGW182 Mediate Binding to hAgo2 (A) Pull-down assays using the ABD of hGW182 as bait were used to capture hAgo. Point mutations of the tandem tryptophans of each of the motifs show similar binding levels as wild-type. A mutant in which all six tryptophans were changed to alanine showed nearly complete loss of binding (rightmost bar). (B) Pull-down assays of hAgo2 with deletion mutants of the ABD of hGW182 with one, two, or all motifs removed. All deletions affected binding to Ago2. Elimination of all three motifs (rightmost bar) had a similar effect on hAgo2 binding to the six-tryptophan point mutant. Data shown are from three different experiments and are shown as average ± SD. (C–H) Analytical SEC of hAgo2-RNA with different deletions of hGW182 ABD. Complexes were formed using a similar pull-down protocol as in (B) and were injected onto a Superdex 200 column. The UV signal at 280 nm was used to monitor elution volumes of the complexes (red) and the individual components: hAgo2-RNA (dashed black line) and ABD (blue). The different ABD deletion constructs used are full-length (residues 455–841) (C), Δ1 (D), Δhook (E), Δ1 and Δ2 (F), Δ1 and Δhook (G), and Δ2 and Δhook (H), and are noted for each chromatogram. See also Figure S4 and Table S2. Molecular Cell 2017 67, 646-658.e3DOI: (10.1016/j.molcel.2017.07.007) Copyright © 2017 Elsevier Inc. Terms and Conditions

Figure 7 hGW182 ABD Can Utilize All Three GW-Binding Motifs to Recruit Multiple Copies of Argonaute (A) MW calibration curve of different hGW182 ABD-hAgo2-RNA complexes. The partitioned coefficient (Kav) was calculated based on the elution volumes presented in Figure 6C and plotted against the predicted log MW of the various complexes. (B) Isothermal titration calorimetry (ITC) analysis of the interaction between hGW182 ABD and three hAgo2 molecules. The ITC cell was filled with hGW182 ABD and hAgo2 was titrated as the ligand. Affinities are reported as dissociation constants (Kd) ± SEs calculated from the fit using the sequential binding model equation. (C) Four selected 2D averages showing three hAgo2s linked together in the presence of hGW182. The number in the lower left corner of each panel refers to the number of raw particles that contributed to the class average. The dimensions of the hAgo2 structure (PDB: 4F3T) were measured in two different orientations (longest and shortest) and used as a reference to estimate the size of each of the subparticles. See also Figure S5. Molecular Cell 2017 67, 646-658.e3DOI: (10.1016/j.molcel.2017.07.007) Copyright © 2017 Elsevier Inc. Terms and Conditions