Conformational Activation of Argonaute by Distinct yet Coordinated Actions of the Hsp70 and Hsp90 Chaperone Systems  Kotaro Tsuboyama, Hisashi Tadakuma, Yukihide Tomari  Molecular Cell  Volume 70, Issue 4, Pages 722-729.e4 (May 2018) DOI: 10.1016/j.molcel.2018.04.010 Copyright © 2018 Elsevier Inc. Terms and Conditions

Molecular Cell 2018 70, 722-729.e4DOI: (10.1016/j.molcel.2018.04.010) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 1 Small RNA Loading Induces Conformational Opening of Ago2 (A) Strategy for N-terminal biotin labeling and site-specific internal double labeling of Ago2. A homology model of fly Ago2 predicted by using human Ago2 (PDB: 4F3T; Elkayam et al., 2012) as the template structure is presented. The same is shown hereafter. (B) Schematic representation of the smFRET analysis of Ago2. (C) Representative smFRET images for apo-Ago2 (upper) and Ago2-RISC (lower). Left (donor) and middle (FRET) panels are presented in a pseudo-color. Donor (green) and FRET (red) images are overlaid in the right panels. Insets show the magnified images of white-framed areas. (D) Density plots of FRET between PAZ- and MID-conjugated dyes for apo-Ago2 (black) and Ago2-RISC (red). FRET efficiency measured for each spot in each frame was accumulated, and the distribution in total effective measurements was plotted. Lines and shadows represent means and SE, respectively, from 3 independent experiments. See also Figures S1 and S2. Molecular Cell 2018 70, 722-729.e4DOI: (10.1016/j.molcel.2018.04.010) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 2 Hsp70+90 Systems Are Sufficient for Conformational Opening of Ago2 (A) Density plots of FRET between PAZ- and MID-conjugated dyes for Ago2 incubated in mock (GST; black), S2 cell lysate (brown), Hsp70+90 systems + Dicer-2/R2D2 (purple), and Hsp70+90 systems alone (green). Lines and shadows represent means and SE, respectively, from 3 independent experiments. (B) Density plots of FRET between PAZ- and MID-conjugated dyes for Ago2 after 10-min (yellow) and 60-min (cyan) incubation in S2 cell lysate with a small RNA. Lines and shadows represent means and SE, respectively, from 2 independent experiments. The red broken line indicates the FRET efficiency for Ago2-RISC, which is the same as the red in Figure 1D. The shift of the FRET peak is not completed even after 60 min, presumably because of the limited accessibility of the surface-tethered Ago2 to small RNAs. Note that Ago2-RISC (the red broken line and Figure 1D) was assembled in solution before surface tethering. (C) Density plots of FRET between PAZ- and MID-conjugated dyes for Ago2 after 10-min (yellow) and 60-min (cyan) incubation in S2 cell lysate without small RNA. Lines and shadows represent means and SE, respectively, from 2 independent experiments. The data for 10-min incubation are the part of data for lysate incubation in (A). See also Figures S2 and S3. Molecular Cell 2018 70, 722-729.e4DOI: (10.1016/j.molcel.2018.04.010) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 3 Hsp70, but Not Hsp90, Partially Populates the Open Form of Ago2 (A) Schematic diagram of the Hsp70/Hsp90 chaperone machinery action, where Hsp40, Hsp70, Hop, Hsp90, and p23 coordinately activate a client protein. (B) Density plots of FRET between PAZ- and MID-conjugated dyes for Ago2 incubated in mock (GST; black), the Hsp70 system (Droj2 and Hsc70-4; blue), the Hsp90 system (Hop, Hsp83, and p23; orange), and the Hsp70+90 systems (green). The plots for mock and Hsp70+90 systems are the same as in Figure 2A. Lines and shadows represent means and SE, respectively, from 3 independent experiments. (C) Small RNA pull-down assay for Ago2 incubated with increasing concentrations of the Hsp70 system, the Hsp90 system, or the Hsp70+90 systems in the presence of the Dicer-2/R2D2 heterodimer. The basal reconstitution system contained 20 nM Dicer-2/R2D2, 150 nM Droj2, 450 nM Hsc70-4, 750 nM Hop, 1.5 μM Hsp83, and 1.5 μM p23, and the concentrations of the Hsp70 system (Droj2 and Hsc70-4) and the Hsp90 system (Hop, Hsp83, and p23) were increased up to 8-fold and those of Hsp70+90 systems were increased up to 4-fold. (D) Quantification of (C). Data represent means ± SD from 4 independent experiments. The p value was calculated by two-sided t test. Molecular Cell 2018 70, 722-729.e4DOI: (10.1016/j.molcel.2018.04.010) Copyright © 2018 Elsevier Inc. Terms and Conditions

Figure 4 Hsp90 System Stabilizes Ago2 in Its Open, Active Conformation (A) Representative traces of FRET (blue line) and ideal path fitting using HMM (red thick line). (B) Donut charts for the accumulated population of HMM-idealized FRET efficiency. “D/R” denotes Dicer-2/R2D2. (C) Transition density plot. The FRET values before (x axis) and after (y axis) each transition were plotted as a two-dimensional chart. Grayscale indicates the ratio of transition events. (D) Boxplots of the dwell time for 4 groups of FRET efficiency. The solid bars indicate median, the boxes indicate the interquartile range (25% and 75%), and the whiskers extend to the most extreme data point no more than 1.5 times the interquartile range from the box. See also Figure S4. Molecular Cell 2018 70, 722-729.e4DOI: (10.1016/j.molcel.2018.04.010) Copyright © 2018 Elsevier Inc. Terms and Conditions