Structural basis for dynamic regulation of the human 26S proteasome

Abstract

The proteasome is the major engine of protein degradation in all eukaryotic cells. At the heart of this machine is a heterohexameric ring of AAA (ATPases associated with diverse cellular activities) proteins that unfolds ubiquitylated target proteins that are concurrently translocated into a proteolytic chamber and degraded into peptides. Using cryoelectron microscopy, we determined a near-atomic-resolution structure of the 2.5-MDa human proteasome in its ground state, as well as subnanometer-resolution structures of the holoenzyme in three alternative conformational states. The substrate-unfolding AAA-ATPase channel is narrowed by 10 inward-facing pore loops arranged into two helices that run in parallel with each other, one hydrophobic in character and the other highly charged. The gate of the core particle was unexpectedly found closed in the ground state and open in only one of the alternative states. Coordinated, stepwise conformational changes of the regulatory particle couple ATP hydrolysis to substrate translocation and regulate gating of the core particle, leading to processive degradation.

Keywords: AAA-ATPase; cyroelectron microscopy; ubiquitin-proteasome system.

Fig. 1.

Cryo-EM structure determination of the human proteasome in four conformational states. (A) A typical cryo-EM micrograph of the human proteasome imaged with a Tecnai Arctica and a Gatan K2 Summit direct detector camera. (B) Typical reference-free 2D class averages of the doubly capped proteasome computed by the ROME software (49). (C) Typical reference-free 2D class averages of the RP–CP subcomplex, showing great detail corresponding to secondary structures of the complex. (D) The cryo-EM density map of the RP–CP subcomplex in a surface representation (Left) and the atomic model built from the density map (Right). (E–G) The cryo-EM densities are shown as solid surfaces for the S_B (E), S_C (F), and S_D (G) states. (H) Representative cryo-EM densities of secondary structures of α-helices in the α3, β3, and β4 subunits (Left three panels) and β-strands in the β3 subunit (Right two panels) in the S_A state are superimposed with the fitted atomic model shown as a stick representation, showing that density quality is sufficient to allow side-chain fitting. The residue numbers of selected bulky side chains are labeled.

Fig. 2.

Conformational changes of the human proteasome in four distinct states. (A) Top view of the lid of S_A. (B–D) Top views of the lid of S_B (B), S_C (C), and S_D (D) superimposed with transparent cartoons of S_A, S_B, and S_C, respectively. (E) Side view of the ATPase ring above the α-ring in S_A. (F–H) Side views of the ATPase ring above the α-ring in S_B (F), S_C (G), and S_D (H) superimposed with transparent cartoons of S_A, S_B, and S_C, respectively. (I) Top view of the ATPase ring in S_A. (J–L) Top views of the ATPase ring in S_B (J), S_C (K), and S_D (L) superimposed with transparent cartoons of S_A, S_B, and S_C, respectively. (M) The α-ring in a cartoon representation from the perspective of the AAA-ATPase or the RP–CP interface in S_A. (N) Close-up view of the central portion of the α-ring, showing that the CP channel is closed in this conformation. The amino-terminal tails of the α2, α3, and α4 subunits blocking the CP channel are shown in stick representation, whereas the rest of the structure is in cartoon representation. (O) The α-ring in a cartoon representation from the perspective of the AAA-ATPase or the RP–CP interface in S_D. (P) Close-up view of the central part of the α-ring, showing that the CP channel is open in this conformation. The amino-terminal tails of the α2, α3, and α4 subunits blocking the CP channel are shown in stick representation, whereas the rest of the structure is in cartoon representation.

Fig. 3.

Structure of the six nucleotide-binding sites of the proteasome. (A) Overview of six nucleotide-binding sites in the AAA-ATPase heterohexamer of the S_A state. Bound nucleotides are shown in stick representation superimposed with cryo-EM densities of the nucleotide that are shown in blue mesh. (B) Close-up views of nucleotide conformations in the six nucleotide-binding sites in S_A. ATP is tentatively modeled into the nucleotide density of each Rpt subunit in S_A.

Fig. 4.

Architecture of the substrate-translocation channel. (A) Overview of the AAA channel calculated by the HOLE program (50). The channel is rendered by surface dots. The dashed green curve indicates the spiral shape formed by the pore-1 loops. (B) Top view of the pore loops aligning along the channel axis from the perspective of the OB domain. (C) Close-up side views of the pore-1 loops from six Rpt subunits decorating the channel, which align along the channel in a spiral staircase formed from Rpt1 to Rpt5, with a backward recession in the Rpt6 pore-1 loop that is slightly away from the major channel pathway. The pore-1 loops form a helical part of the channel interior as illustrated by the dashed green line. (D) Close-up side view of the pore-2 loops from six Rpt subunits decorating the channel, which form a complete spiral staircase from Rpt1 to Rpt6. These pore-2 loops form another helical part of the channel interior, illustrated by the red dashed line. (E) Side view of the complete ATPase channel, including components from both the OB and AAA domains, calculated by the HOLE program (50). Side-chain patterns observed along the substrate-translocation pathway are highlighted. The five tyrosine residues, highlighted by transparent sphere representation, and a number of hydrophobic and negatively charged residues decorate the AAA channel; color codes for ATPase protomers match those shown in A. (Inset) A schematic cartoon showing that the pore-2 loops of Rpt3, Rpt4, and Rpt5 pair laterally with the pore-1 loops of Rpt4, Rpt5, and Rpt1, respectively, to form the three narrowest constrictions in the AAA channel. (F) The channel radius along the pore axis approximately estimated by HOLE (50), showing the three narrowest constrictions in the AAA channel but only one narrow constriction in the OB channel that is more than twice as wide as those of the AAA channel.

Fig. 5.

RP–CP interface regulates gating of the CP channel. (A) Overview of the RP–CP interface in which the carboxyl-terminal tails of Rpt3 and Rpt5 are shown to insert into the α-pockets in the S_A state. Rpt6 is not shown, for clarity. The dashed circles mark the HbYX motifs of two Rpt subunits that insert into the α-pockets. (B) Overview of the RP–CP interface in which the carboxyl-terminal tails of Rpt1, Rpt2, Rpt6, Rpt3, and Rpt5 are shown to insert into the α-pockets in the S_D state. The dashed circles mark the HbYX motifs of five Rpt subunits that insert into the α-pockets. (A and B, Insets) Simplified illustrations of the tail–pocket interactions between the ATPase ring and CP. The orange hexagons and blue heptagons represent the ATPase and the α-ring, respectively. The small circles connected to the orange hexagon represent the carboxyl-terminal tails of the Rpt subunits inserted into the α-pockets. (C) Close-up view of interactions between the C-terminal HbYX motifs of Rpt3 and the α1–α2 pocket in the S_A state. (D) Close-up view of interactions between the C-terminal HbYX motifs of Rpt5 and the α5–α6 pocket in the S_A state. (E–G) Close-up view of interactions between the Rpt1 (E), Rpt2 (F), and Rpt6 (G) C termini and the α4–α5, α3–α4, and α2–α3 pockets in the S_D state, respectively. (H) Hypothetical structure-based model for substrate degradation by the human proteasome. UIM, ubiquitin-interacting motif.