Fig 1: RRP1B binds PP1 via multiple interaction surfaces(A) Crystal structure of the RRP1B682–727:Q20RPP1α7–300 complex. RRP1B is shown as yellow sticks with PP1 shown as a gray surface. PP1 SLiM binding pockets to which RRP1B binds (RVxF, ΦΦ[xF], SILV, AF, SILK) are colored from teal to coral. Dashed boxes correspond to the zoomed-in pockets shown in (D), (E), (G), (H), and (I).(B) Upper: RRP1B sequence with secondary structure (top) and SLiM motifs (bottom) indicated. Lower: percentage of buried surface area plotted by residue for each RRP1B chain (two complexes were present in the asymmetric unit). Shaded areas correspond to RRP1B residues that bind in PP1 SLiM binding pockets and colored according to shaded pockets in (A). Gray shaded box highlights RRP1B residues that adopt distinct conformations in the two complexes.(C) Sequence logo of RRP1B sequences from 319 distinct species; sequence crystallized is colored gray.(D and E) Major binding interactions between RRP1B (yellow sticks) and PP1 (surface), colored as in (A). The well-established SLiM binding pockets (D, RVxF; E, ΦΦ[xF]) are shown.(F) Overlay of the RVxF and ΦΦ[xF] structures from nine PP1 holoenzymes, with residues binding RVxF, ΦΦ, and ΦΦ[xF] pockets shown as sticks.(G–I) SILV (L704 sidechain not shown for clarity, G), AF (H), and SILK (I) binding pockets with RRP1B (yellow sticks) and PP1 (surface), colored as in (A).(J) Both RRP1B:PP1 complexes present in the asymmetric unit overlaid using PP1; the distinct RRP1B chains are colored yellow and blue.
Fig 2: Interaction of RRP1B with PP1 isoforms(A and B) ITC thermograms of RRP1B657–758 with PP1γ (A) or PP1α (B).(C) Localization of GFP and GFP-PP1γ constructs in live U2OS cells counterstained with the permeable DNA dye Hoechst 33342 (representative images from three biological replicates). Scale bars, 5 μm.(D) Western blot analysis of co-precipitation of ASPP2 and RRP1B with GFP and GFP-PP1γ constructs captured on GFP-Trap_A beads.(E) ITC thermogram of RRP1B657–758 with PP1α7–330 Q20R.(F) Localization of GFP-PP1α, GFP-PP1γ, GFP-PP1α Q20R, and GFP-PP1γ R20Q constructs in live U2OS cells counterstained with the permeable DNA dye Hoechst 33342 (representative images from three biological replicates). Scale bars, 5 μm.(G) Western blot analysis of co-precipitation of RepoMan and RRP1B with GFP, GFP-PP1α, GFP-PP1γ, GFP-PP1α Q20R, and GFP-PP1γ R20Q constructs captured on GFP-Trap_A beads.(H) Quantification of RRP1B co-precipitated by the indicated PP1 variants (D and G), relative to WT (mean ± SE, n ≥ 3). For each experiment, Image Lab software was used to quantify the RRP1B signal per GFP signal.See also Table S1 and Figure S1.
Fig 3: RRP1B phosphorylation weakens PP1 binding(A–C) The Arg20 β/γ-specificity pocket with the PP1 surface colored by electrostatics and RRP1B (A: Ser702) or modeled RRP1B variants (B: S702D; C: pSer702) shown as sticks (yellow); side-chain atoms are shown as dots to illustrate their space-filling volume.(D) SPR sensorgram between RRP1BS702D and PP1α7–330 Q20R.(E–H) HeLa/TetU2 cells with stably integrated 256xLacO repeats transiently transfected with pmCherry-LacR-NLS-PP1γ and either GFP-RRP1B WT (E), GFP alone (F), GFP-RRP1BRAxA (G), or GFP-RRP1BS702D (H). In four biological replicates (representative images shown here), GFP-RRP1B WT accumulated at the gene locus in 98% ± 2% of 120 cells scored, GFP alone and GFP-RRP1BRAxA in 0% of 120 cells scored, and GFP-RRP1B S702D in 7% ± 2% of 120 cells scored. Graphs illustrate plot line profiles for red and green fluorescence intensity over 2 μm at the gene locus. Scale bars, 5 μm.(I) Western blot analysis of co-precipitation of PP1γ from U2OS cells with GFP-RRP1B, GFP-RRP1BRAxA, and GFP-RRP1BS702D (representative blot from three biological replicates).(J) Quantification of PP1γ co-precipitated by the indicated RRP1B variants (I), relative to WT GFP-RRP1B (mean ± SE; n ≥ 3). For each experiment, Image Lab software was used to quantify the PP1γ signal per GFP signal. The amount of PP1γ pulled down by GFP-RRP1BRAxA is 2.1% ± 0.6% and that of GFP-RRP1BS702D is 4.6% ± 2.0% compared with GFP-RRP1B WT.See also Tables S3 and S4 and Figure S3.
Fig 4: IDP regulators that are specific for PP1β/PP1γ bind the β/γ-specificity pocket(A) Overlap of the RRP1B:PP1, Ki-67:PP1, and RepoMan (RM):PP1 complexes. RRP1B (yellow), Ki-67 (cyan), and RM (blue) are shown as ribbons with PP1 shown as a gray surface. The β/γ-specificity pocket is magenta. Side chains that bind the major interaction pockets are shown as sticks.(B) Structure-based sequence alignment of the PP1 interaction domains of RRP1B, Ki-67, and RM, with PP1 interacting residues indicated. Underlined residues have identical conformations.(C) Close-up view of the interactions stabilizing the loops between the [xF] Phe residue and the β/γ-specificity pocket binding residue. Regulators are colored as in (A) and shown as sticks. Side chains are shown for the Phe and β/γ-specificity pocket binding residue.(D–F) Same view as (C), but on the RRP1B:PP1 (D), RM:PP1 (E), or Ki-67:PP1 (F) complexes. Hydrogen bonds are indicated by dashed lines, with the distances in angstroms.(G) Cartoon highlighting the major regulators of PP1γ in the nucleolus.(H) RRP1B:PP1 holoenzyme assembly is regulated by phosphorylation on a least two residues, Thr685 (the “x” residue in the RRP1B RVxF motif) and Ser702. Potential kinases are indicated (phosphosite3.1);40 the reversing phosphatases are unknown.
Fig 5: Non-canonical regulator interaction sites on PP1(A) The Arg20 β/γ-specificity pocket. Left; PP1α Q20R is shown as an electrostatic surface while RRP1B (yellow) is shown as a ribbon with a subset of side chains, including Ser702, shown as sticks. Arg20 β/γ-specificity pocket is highlighted by a dashed line (cyan). Right: same view, but with PP1 (white, RRP1B-bound PP1; magenta, free PP1) and RRP1B (yellow) shown as sticks. Hydrogen bonds/salt bridges are shown as black dashed lines. Arrows highlight the rotations of PP1 Arg74 and Glu77 that occur upon RRP1B binding. The location of the Arg20 β/γ-specificity pocket is indicated in gray with a cyan dashed line. PP1 residues underlined correspond to the PP1 N-terminal loop.(B) The N-terminal binding groove. Left: PP1 is shown as an electrostatic surface while RRP1B (yellow) is shown as a ribbon with a subset of side chains, including Ser702, shown as sticks. The N-terminal groove is highlighted by a dashed line (cyan). Right: same view, but with PP1 (white) and RRP1B (yellow) shown as sticks. Hydrogen bonds/salt bridges are shown as black dashed lines. The location of the N-terminal binding groove is indicated in gray with a cyan dashed line. PP1 residues underlined correspond to the PP1 helix α1.(C–E) SPR sensorgrams between RRP1B (WT and variants) and PP1α7–330 Q20R. (C) WT RRP1B682–727, (D) RRP1B682–727 F715A, (E) ΔSILK: RRP1B682–723, and (F) RRP1B682–727 KATA (RAxA mutant).See also Table S3.
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