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Review
. 2018 Jun 20:87:697-724.
doi: 10.1146/annurev-biochem-062917-011931. Epub 2018 Apr 13.

Structure and Function of the 26S Proteasome

Jared A M Bard  1   2 , Ellen A Goodall  1   2 , Eric R Greene  1   2 , Erik Jonsson  1   2   3 , Ken C Dong  1   2   3 , Andreas Martin  1   2   3
Affiliations
Review

Structure and Function of the 26S Proteasome

Jared A M Bard et al. Annu Rev Biochem. .

Abstract

As the endpoint for the ubiquitin-proteasome system, the 26S proteasome is the principal proteolytic machine responsible for regulated protein degradation in eukaryotic cells. The proteasome's cellular functions range from general protein homeostasis and stress response to the control of vital processes such as cell division and signal transduction. To reliably process all the proteins presented to it in the complex cellular environment, the proteasome must combine high promiscuity with exceptional substrate selectivity. Recent structural and biochemical studies have shed new light on the many steps involved in proteasomal substrate processing, including recognition, deubiquitination, and ATP-driven translocation and unfolding. In addition, these studies revealed a complex conformational landscape that ensures proper substrate selection before the proteasome commits to processive degradation. These advances in our understanding of the proteasome's intricate machinery set the stage for future studies on how the proteasome functions as a major regulator of the eukaryotic proteome.

Keywords: 26S proteasome; AAA+ ATPase; deubiquitination; energy-dependent protein degradation; ubiquitin code; ubiquitin receptor.

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Figures

Figure 1
Figure 1
Structure and conformational changes of the proteasome. (a) The 26S proteasome is composed of three subcomplexes: the core (gray); the base (with Rpn2 and the motor subunits Rpt1–Rpt6 in light blue and the ubiquitin-binding subunits Rpn1 and Rpn13 in dark blue); and the lid (with Rpn3, Rpn5, Rpn6, Rpn7, Rpn8, Rpn9, Rpn12, and Sem1 in yellow, and the DUB Rpn11 in orange). The ubiquitin receptor Rpn10 is shown together with the lid (dark blue). (Left) The three subcomplexes are depicted individually; (center and right) the entire 26S proteasome structure is shown (EMDB: 3534) (40). The center orientation allows a view of the entrance to the central pore and the Rpn11 active site, and the right orientation, rotated by 120°, emphasizes the lid subcomplex with its hand-shaped structure of the PCI (proteasome-CSN-initiation factor 3) domain-containing subunits. (b) Conformational switching of the 19S regulatory particle between the s1 state (EMDB: 3534) and the s3 state (EMDB: 3536) (40), with the core particles aligned. Shown are the views from the right and back of the proteasome relative to the center orientation in panel a. In the s1 conformer, the Rpt ring and Rpn2 are depicted in light blue; Rpn1, Rpn10, and Rpn13 in cyan; and the lid in yellow. In the s3 conformer, the Rpt ring and Rpn2 are depicted in medium blue; Rpn1, Rpn10, and Rpn13 in dark blue; and the lid in salmon. For both conformers, the core is shown in gray. During the transition from s1 to s3, the lid and Rpn10 rotate by ~30° relative to the Rpts. (c) Cutaway representations of the proteasome in the conformations s1–s4, emphasizing differences in the location of Rpn11; the width of the central processing channel; and the coaxial alignment of the N-ring, the AAA+ (ATPases associated with various cellular activities) ring, and the 20S core. The central channels through the N-ring and AAA+ ring are highlighted by a solid black line. The coaxial alignment is most pronounced in the s3 and s4 conformers, leading to the formation of a wide continuous channel for substrate translocation, with the Rpn11 active site (red dot) located directly above the entrance. Also shown are top-down views of the 20S core particle, emphasizing the changes in the 20S gate, which has the most density in the s1 state and the least in the s4 state.
Figure 2
Figure 2
Model for substrate engagement by the proteasome. (Left) Proteasome is shown in the s1 state (EMDB: 3534) with a model substrate (red) tethered through a tetraubiquitin chain (purple) near the presumed location of the Rpn10 ubiquitin-interacting motif. In this s1 state, Rpn11 (orange) is offset to the right, making the entrance to the central pore accessible for insertion of a substrate’s unstructured initiation region. (Right) Substrate engagement shifts the proteasome to the s3 or s4 state (s4 is shown; EMDB: 3537) (40) in which the N-ring and AAA+ (ATPases associated with various cellular activities) ring are coaxially aligned to facilitate substrate translocation and the 20S gate is open for polypeptide transfer into the internal degradation chamber. Rpn11 is located directly above the entrance to the central pore, where it acts as a gatekeeper and removes ubiquitin chains from substrates during translocation. The initiation region of the substrate must be long enough to bridge the gap between the N-ring and the pore loops of the Rpts.
Figure 3
Figure 3
Conformational landscape of the proteasome. (a) Qualitative comparison of how the presence of nonhydrolyzable ATP analogs, protein substrate, and ubiquitin-bound Ubp6 affects individual enzymatic activities of the proteasome. Up and down arrows indicate the stimulation and inhibition, respectively, of enzymatic activities of the proteasome compared with their basal activities; two up arrows indicate hyperstimulation. Basal activities are defined here for the proteasome in the presence of ATP and absence of substrate proteins. (b) Data from 17 electron microscopy data sets that directly compare the relative abundances of proteasome conformations under various conditions. The data sets are clustered according to the experimental conditions. Increased ATPase and core-peptidase activities appear to be correlated with greater abundance of s3 and s4 states. (Right) Resolutions of all structures obtained, representing the FSC value at 0.143. An asterisk indicates resolution was attained with an FSC value of 0.3; double asterisks indicate resolution was attained with an FSC value of 0.5. Abbreviations: FSC, Fourier shell correlation; NA, not applicable; NR, not reported; Ub, ubiquitin.
Figure 4
Figure 4
AAA+ motor architecture, nucleotide binding, and conformational changes. (a) Side (left) and top (right) views of the EM density for the Rpt1–Rpt6 hexamer in the s3 state (EMDB: 3536) (40). The small AAA+ subdomain of each Rpt forms a rigid body with the large AAA+ subdomain of the clockwise-next neighboring subunit. The rigid body formed between Rpt3 (red) and Rpt6 (dark blue) is shown with a dashed loop. In the top view (right), the EM densities for Rpt3 and Rpt6 are shown in transparent grey, and the molecular models for both Rpts are fitted into the density, with the large AAA+ subdomain of Rpt3 in red and the small AAA+ subdomain of Rpt6 in blue, to highlight the rigid-body interaction. (b, left) Molecular model of Rpt3 (PDB: 5mpb) (40); (right) representation of the relative positions of the large and small AAA+ subdomains as well as the pore loop responsible for mechanical substrate translocation. (c) Schematic of the Rpt1–Rpt6 hexamer, showing the reported nucleotide occupancy of each AAA+ binding pocket for EM reconstructions in the presence of ATP (s1 state) (innermost circle in dark gray) or different ATP analogs (ADP-BeFx, ADP-AlFx, and two distinct ATPγS structures): Black dots indicate the presence of nucleotide density, dots are absent from reported empty sites, and outlined gray dots denote sites with lower probability occupancy or lower affinity. For the s1 state, all available data sets show nucleotide density in every pocket (white dots), although Rpt6 exhibits a smaller density and a lack of arginine-finger contacts, suggesting that the bound nucleotide is ADP (white star) (40, 43, 44, 50, 51). The two ATPγS data sets reflect the higher-certainty assignments from Zhu et al. (45), the results for ADP-AlF4-bound proteasomes were taken from Ding et al. (43), and the ADP-BeFx data set reflects the assignments of the s4 state described in Wehmer et al. (40). (d) Representations of the splayed-out Rpt subunits in the steep spiral arrangement of the s1 state and the more planar staircase conformation in s3. All subunits are oriented with the channel-facing pore loops pointing to the right. The dashed lines serve to highlight the apparent tilt of the AAA+ large domains. Abbreviations: AAA+, ATPases associated with various cellular activities; EM, electron microscopy; OB, oligonucleotide/oligosaccharide binding.
Figure 5
Figure 5
Ubiquitin receptors and DUBs (deubiquitinases) at the proteasome. (a) Cryo-electron microscopy (EM) reconstruction of the 26S proteasome in complex with ubiquitin-bound Ubp6 (EM: 3034; PDB: 5a5b) (56), with EM density for the lid shown in yellow, the base in light blue, the core particle in gray, Rpn11 in orange, Ubp6 in lighter orange, and individual ubiquitin receptors in darker blue. A schematic version highlighting the DUBs and ubiquitin receptors labeled by figure panel is shown in the inset (lower right). The essential DUB Rpn11 (orange) sits just above the N-ring pore with its active site 35 Å away from the active site of Ubp6 (lighter orange, with bound ubiquitin density in pink). Bridging EM density links Ubp6’s ATPase-contacting ubiquitin-specific protease (USP) domain to the UBL (ubiquitin-like) domain bound at the T2 site of Rpn1. The UBL domain of Mus musculus Ubp6 (PDB: 1wgg) (orange ribbon) is fit into the EM density seen at the T2 site of Rpn1, using Chimera’s Fit in Map tool. The USP domain of Ubp6 (PDB: 5a5b) is also depicted as an orange ribbon within the EM density. Each ubiquitin receptor on the proteasome is shown with the ribbon diagram of the EM-based atomic model docked into the EM density. Proteasome-bound ubiquitin (pink ribbon) is modeled by docking existing ubiquitin-receptor costructures into the EM density for each ubiquitin receptor. K48-linked diubiquitin bound to the dual UIMs (ubiquitin-interacting motifs) of human S5a/PSMD4 (Rpn10) is shown in a possible location on the proteasome, placed by confining the most N-terminal residue of the UIM structure 17 Å from the most C-terminal residue of the EM docked Rpn10 VWA (von Willebrand factor type A) domain. This constraint is determined by the five amino acid linker unresolved between the UIM and VWA domains in the available structures of the Homo sapiens Rpn10 homolog (PDB: 2kde) (134). (b) The side view of the human S5a/PSMD4 (Rpn10) with bound diubiquitin highlights the gap (dashed line) between the VWA domain and the UIMs. In organisms containing more than a single UIM in Rpn10, UBLs such as hHR23 (dark green) can bind to the UIM2 site in a similar manner as ubiquitin, as illustrated by overlaying the hHR23 S5a structure with the ubiquitin dimer-bound structure of S5a (PDB: 1p9d) (173). Here, the C terminus of ubiquitin (pink sphere) is shown 76 Å from the active site of Rpn11, but this distance is only a single possibility owing to the flexibility in the linker between the UIMs and the VWA domain anchoring Rpn10 to the proteasome. (c) The T1 site of Rpn1 is shown bound to the K48-linked ubiquitin dimer, with the free ubiquitin C terminus (pink sphere) sitting 93 Å from the active site of Rpn11 (distance indicated as green dashed line) (PDB: 2n3v) (18). Side view compares the interaction of UBLs or ubiquitin with Rpn1. Rad23 (dark green) interacts with the portion of the T1 Rpn1 site that is bound by the distal ubiquitin (the ubiquitin without a free C terminus in the dimer) in the ubiquitin dimer-bound structure (PDB: 2nbw) (131). The free C terminus of ubiquitin is highlighted with a pink sphere, whereas the most C-terminal residue of Rad23 is highlighted with a smaller green sphere. The M. musculus Ubp6 UBL is placed at the T2 site, which is distinct from the T1 ubiquitin binding site. The structure of the human Rpn13 homolog ADRM1 (dark blue) (PDB: 5v1z) (174) in complex with ubiquitin and the respective Rpn2 peptide is placed into the EM density, using the orientation defined by Wehmer et al. (40) (PDB: 5mpd). This orientation places the C terminus of ubiquitin 91 Å from the active site of Rpn11. (d) The UBL of Dsk2 binds to Rpn13 in a similar manner as ubiquitin, highlighted by the overlay of the ADRM1 bound to hPLIC2, a Dsk2 homolog (dark green) (PDB: 2nbv) (131), with the C-terminal residues indicated by same-colored spheres.
Figure 6
Figure 6
Crystal structures of proteasomal DUBs. (a) Crystal structure of Saccharomyces cerevisiae Rpn11 (orange) bound to ubiquitin (green) (PDB: 5u4p) (42) and docked into the electron microscopy density with Ubp6 removed for clarity (EM: 3034; PDB: 5a5b) (56). The central pore is highlighted by a red sphere. (b) Arrangement of the Ins-1 loop of Rpn11 (cyan) in (top) the unbound apo state (PDB: 4o8x) (155) and in (bottom) the ubiquitin-bound state (PDB: 5u4p) (42), with the C-terminal tail of ubiquitin in green. The catalytic zinc ion (gray sphere) is shown coordinated by the catalytic residues (stick representation). (c) Crystal structure of activated Uch37 (purple) bound to the DEUBAD of Rpn13 (yellow) and ubiquitin (green) (PDB: 4wlr) (168). Abbreviations: DEUBAD, deubiquitinase adaptor; DUB, deubiquitinase; Ins-1, Insert-1; Ub, ubiquitin.
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