Characterization of Fully Recombinant Human 20S and 20S-PA200 Proteasome Complexes

Abstract

Proteasomes are essential in all eukaryotic cells. However, their function and regulation remain considerably elusive, particularly those of less abundant variants. We demonstrate the human 20S proteasome recombinant assembly and confirmed the recombinant complex integrity biochemically and with a 2.6 Å resolution cryo-EM map. To assess its competence to form higher-order assemblies, we prepared and analyzed recombinant human 20S-PA200, a poorly characterized nuclear complex. Its 3.0 Å resolution cryo-EM structure reveals the PA200 unique architecture; the details of its intricate interactions with the proteasome, resulting in unparalleled proteasome α ring rearrangements; and the molecular basis for PA200 allosteric modulation of the proteasome active sites. Non-protein cryo-EM densities could be assigned to PA200-bound inositol phosphates, and we speculate regarding their functional role. Here we open extensive opportunities to study the fundamental properties of the diverse and distinct eukaryotic proteasome variants and to improve proteasome targeting under different therapeutic conditions.

Keywords: 20S-PA200; PA200; atomic model; cryo-EM; human; proteasome; recombinant; regulator; structure.

Graphical abstract

Figure 1

Biochemical Characterization of Recombinant Human 20S Proteasome and 20S-PA200 Complexes (A) SDS-PAGE of endogenous (e20S) and recombinant (r20S) human 20S proteasomes and recombinant human 20S-PA200 complexes (r20S-PA200) (see also Figures S1A and S1B). (B) Proteolytic activities of e20S, r20S, and r20S-PA200 against the fluorogenic substrates Z-LLE-AMC (blue), Boc-LRR-AMC (magenta), and Suc-LLVY-AMC (red), specific for the proteasome β1 caspase-like, β2 trypsin-like, and β5 chymotrypsin-like active sites, respectively. Similar 20S proteasome molarities were used in each assay, as shown in (A). (C) Proteolytic activities of recombinant human 20S proteasomes in the presence of increasing concentrations of PA200, color coded as in (B) (see also Figure S1C). Error bars are represented as mean ± SD.

Figure 2

Cryo-EM Structures of the Recombinant Human 20S Proteasome and 20S-PA200 Complex (A) Cryo-EM structure of the recombinant human 20S proteasome with a fitted atomic model (see also Figure S2). (B and C) Two close-up views of the structure shown in (A), which has well-resolved side chains throughout. (D) Cryo-EM structure of the recombinant human 20S-PA200 complex with a fitted atomic model (see also Figures S2 and S3). (E and F) Two close-up views of the structure shown in (D), which has well-resolved side chains throughout. (G) Overall view of the 20S-PA200 atomic model, with major domains indicated. (H) Close-up views of the 20S-PA200 cryo-EM map (gray mesh) with a fitted atomic model (cartoon representation), with each subunit color-coded as indicated at the top. In (A)–(F) The cryo-EM maps are shown as mesh and the protein models as cartoons (A and D) or sticks (B, C, E, and F).

Figure 3

PA200 Main Docking Sites on the Proteasome α Rings (A) Cartoon representation of the PA200 structure, showing the two major anchor regions of PA200 at the proteasome α ring, one involving the PA200 loop formed by residues 561–576 (dashed circle, left) and the other involving the PA200 C terminus (solid circle, right). (B) Close-up view of interactions between the PA200 loop, residues 561–576, and the proteasome subunits α1 (orange) and α2 (green). (C) Close-up view of interactions between the PA200 C terminus and the proteasome subunits α5 (blue) and α6 (red). In (A)–(C) the 20S-PA200 structure is oriented to best depict the interactions highlighted. In (B) and (C), the cryo-EM maps are shown as gray mesh and the atomic models as sticks. The protein-protein interaction network involving these two PA200 anchor regions and the 20S proteasome are represented in Figures S4A and S4B. See also Figure S5.

Figure 4

PA200 Induced Conformational Changes in the 20S Proteasome α Rings (A) The closed α ring outer surface of the 20S proteasome. (B) Superimposition of the α ring outer-surface atomic models of the recombinant human 20S proteasome (gray cartoon) and 20S-PA200 (cartoon with subunits color coded). The H0 helices of the 20S proteasome α1–α2 and α4–α7 are indicated by solid circles color coded as in 20S-PA200, whereas the H0 helix of α3, which is disordered in 20S-PA200, is encircled by a black dashed circle. (C) The open α ring outer surface of the 20S-PA200 complex. In (A) and (C), the cryo-EM maps are shown as gray mesh, and the atomic models are represented as cartoons.

Figure 5

Interaction between the N-Terminal Loops of the Proteasome α Subunits with PA200 (A) Cartoon representation of the N-terminal tails of the proteasome α5–α7 subunits, indicated by arrows, at the PA200 dome inner surface. (B–D) Close-up views of the N termini of the proteasome subunits α7 (B), α6 (C), and α5 (D). The cryo-EM maps are shown as gray mesh and the atomic models as sticks. In (A)–(D), the 20S-PA200 structure is oriented to best depict the highlighted interactions. The protein-protein interaction network involving the N-terminal tails of the proteasome α5–α7 subunits and PA200 are represented in Figures S4C–S4E.

Figure 6

Relevant Features in the Structure of the Human PA200 (A) van der Waals surface representation, colored by charge, of the PA200 distal outer surface (oriented as indicated on the left), showing two prominent positively charged grooves, indicated by solid rings. (B) Close-up view of the non-protein density blocking the channel on the positively charged groove indicated in (A). (C) The densities for the cofactor in (B) are fitted with a model of InsP6. Only one of the InsP6 phosphate groups does not interact directly with PA200 and, consequently, is somewhat less well recovered in our map. (D) The location of InsP6 (B and C) within its PA200 groove. (E) Close-up view of the non-protein density blocking the channel of the positively charged groove indicated in (A). (F) The densities for the cofactor in (E) are fitted with a model of 5,6[PP]₂-InsP4. (G) The location of 5,6[PP]₂-InsP4 within its PA200 groove. See also Figures S5 and S6.

Figure 7

Comparison of the Proteolytic Active Sites in the Human 20S Proteasome and 20S-PA200 Complexes Shown are van der Waals surface representations, colored by charge, of the three proteasome active sites (β1, β2, and β5) of the recombinant 20S proteasome (top row) and 20S-PA200 complexes (bottom row), viewed from the proteasome inner cavity. White dashed circles indicate the S1 pocket of each active site.