Fig 1.
NS-TEM images and cryo-EM structures of human PA200 and PA200-20S proteasome.
(A) PA200 and 20S were incubated at the ratio of 4.4:1 at room temperature for 1 h and then imaged by NS-TEM. Different particles were classified by 2D class averages and counted. Of the complexes, 33.3% (4,119 out of 12,363) were PA2002-20S (double-capped), and 51.0% (6,305 out of 12,363) of the complexes were PA200-20S (single-capped). (B) Composition of three different particles at the PA200:20S ratio of 8.8:1. PA2002-20S and PA200-20S were 47.6% (7,642 out of 16,049) and 40.2% (6,455 out of 16,409) in the complex. (C) Representative micrographs of frozen-hydrated PA200-20S complex particles. (D) Final cryo-EM density map of PA200-20S colored according to the local resolution. (E) Representative 2D class averages in different orientations of the complex. (F) Gold-standard FSC curves of PA200-20S complex. The resolution was determined to be 2.72 Å, and the 0.5 cutoff value is indicated by a horizontal yellow dashed line. (G) Representative micrographs of frozen-hydrated PA200 particles. (H) Final cryo-EM map of PA200 colored according to the local resolution. (I) Representative 2D class averages in different orientations of PA200. (J) Gold-standard FSC curves of PA200. The resolution was determined to be 3.75 Å, and the 0.5 cutoff value is indicated by a horizontal yellow dashed line. cryo-EM, cryo–electron microscopy; FSC, Fourier shell correlation; NS-TEM, negative-stain transmission electron microscopy; PA200, proteasome activator 200.
Fig 2.
The overall structures of PA200 and PA200-20S.
(A) PA200 (cylindrical helices) forms a largely closed dome-like shape as seen from the top view. The N-terminal is indicated with a black arrow. (B) Same as (A) but the dome is rotated 180° to show the bottom view. (C) Structural alignment of PA200 (PDB: 6KWY) and Blm10 (PDB: 4V7O), shown as top view. Blm10 (green) is slightly bigger than the dome of PA200 (magenta). (D) Bottom view of the structural alignment of PA200 and Blm10. (E) Side view, space-filling representation of PA200-20S complex. PA200 is shown as magenta, and the subunits of the 20S are shown in different colors. (F) Same as (E) but the complex is rotated 90° and with a lateral chip from this perspective. The top subunits α1, α6, and α7 have smaller volume interaction with PA200. (G) The overall structure of PA200 (gray) from the PA200-20S complex structurally aligned with unbound PA200 (magenta). (H) Same as (G) but the dome is rotated 90° to show the side view. Blm10, Bleomycin resistance 10; PA200, proteasome activator 200; PDB, Protein Data Bank.
Fig 3.
Gate opening and the peptidase activity of the 20S induced by PA200.
(A) After the combination of PA200, the gate of the 20S formed by top α subunits became disordered (shown within a dotted circle). (B) Close-up views of the top α-rings. The cryo-EM maps are shown as gray mesh and the atomic models as a cartoon. The N-terminals of the top α subunits became disorder and invisible. (C) The bottom gate of the 20S is closed because PA200 is not bound to it to induce gate opening. The N-terminals of all seven α-subunits point to the center of the hole and close the gate. (D) Close-up views of the bottom α-rings. The cryo-EM map (gray mesh) with a fitted atomic model (cartoon representation). Unlike panel B, the N-terminals of the bottom α subunits point to the central of the closed gate. (E) A surface cut of density map gives a clear sectional view with the open gate (green arrow) on top, closed bottom gate (black arrow), and opening 1 with the density map of the cofactor (red arrow). (F) The other cutting angle of the sectional view gives further proof of the two gates and the opening 2 with its cofactor map. (G) The proteasome activity of the 20S and PA200-20S was evaluated by the 20S proteasome assay kit, and Suc-LLVY-AMC was used as substrate. First, 20S CP and PA200-20S CP (2.5 nM) were incubated with 100 μM Suc-LLVY-AMC for 15 min at 25°C, and then fluorescence measurements (RFU) were taken at 30-s intervals and plotted against time. Data underlying these plots for top panel can be found in S1 Data. The chymotryptic-like activity of 20S and 20S-PA200 were analyzed at different concentrations of Suc-LLVY-AMC (25, 50, 100, 150, 200, 300 μM for 20S and 25, 50, 100, 200, 300, 400, 500 μM for 20S-PA200). The underlying numerical data and statistical analysis for middle and bottom panels can be found in S2 and S3 Data. CP, core particle; cryo-EM, cryo–electron microscopy; PA200, proteasome activator 200; RFU, relative fluorescence units.
Fig 4.
Interactions between PA200 and 20S.
(A) Five important parts involved in the interactions are shown in the black dashed box after a little movement of PA200 apart from the 20S. (B-F) Close-up views of contact areas YYA (dark green) (B), loop (562–572, green) (C), α5 (N-terminal of subunit α5, lemon) (D), α6 (N-terminal of subunit α6, cyan) (E), and α7 (N-terminal of subunit α7, gray) (F). Key residues involved in interactions between PA200 and 20S are labeled and shown as sticks. The hydrogen bonds are shown as dashed lines. PA200, proteasome activator 200.
Fig 5.
Structural details of the two openings and the cofactors bound by PA200.
(A-B) Close-up view of the openings on Blm10 (PDB: 4V7O) (A) and PA200 (B) dome. The location on PA200 corresponding to the largest opening of Blm10 is covered by two loops (loop3, blue; and loop4, red). Residues on the two loops involved in the interaction with other nearby residues are shown as sticks, and the hydrogen bonds are shown as dashed lines. Two regions are missing in the previously reported Blm10 structure (L154–N239 and Y1037–L1147). However, these two regions happen to be the reported Blm10 opening. (C) PA200 is shown as electrostatic surface, and the two openings are shown in black circles. (D) Close-up views of the two openings on PA200 and its cofactors. Positively charged residues located at the openings and involved in the interaction are shown as sticks indicated in different colors (cyan sticks in opening 1 and yellow sticks in opening 2). The electron densities bound in opening 1 and 2 are compatible with 5,6[PP]2-InsP4 and InsP6, respectively. The dimensions of the two openings are 19.6 × 13.7 Å and 23.3 × 17.8 Å, respectively. 5,6[PP]2-InsP4, (5,6)-bisdiphosphoinositol tetrakisphosphate; Blm10, Bleomycin resistance 10; InsP6, inositol hexakisphosphate; PA200, proteasome activator 200.
Fig 6.
Identification of 5,6[PP]2-InsP4 and InsP6 by HPLC-MS.
(A) The InsP6 standard (phytic acid sodium salt hydrate, C6H18O24P6·xNa+·yH2O) was analyzed by HPLC-MS system. The main characteristic MS peak at 814.85, corresponding to (MInsP6+7Na)+. The molar mass of InsP6 is 660.029 g·mol−1. (B) PA200 was validated using the same program, and the MS gave one characteristic peak at 814.92 corresponding to (MInsP6+7Na)+ (red) and a series of peaks that represent different forms of sodium 5,6[PP]2-InsP4 with various number of Na+ ions, i.e., peak 1,019.19 (M5,6[PP]2-InsP4+9Na)+ (cyan), peak 1,041.13 (M5,6[PP]2-InsP4+10Na)+ (green), and peak 1,063.24 (M5,6[PP]2-InsP4+11Na)+ (blue). 5,6[PP]2-InsP4, (5,6)-bisdiphosphoinositol tetrakisphosphate; HPLC-MS, high-performance liquid chromatography–mass spectrometry; InsP6, inositol hexakisphosphate; PA200, proteasome activator 200.
Fig 7.
(A-B) Ribbon representation of the PA200 BRDL domain in top view (A) and side view (B), with the key residues shown as sticks. (C) The alignment of the PA200 BRDL domain (magenta) and the corresponding region (cyan) of Blm10. The region corresponding to BRDL domain of Blm10 is shown as yellow. The N-terminal of Blm10 is shown as red spheres, whereas the C-terminal is indicated as blue spheres (see also S4 Fig). Blm10, Bleomycin resistance 10; BRDL, bromodomain-like; PA200, proteasome activator 200.