Molecular architecture of the 26S proteasome holocomplex determined by an integrative approach

Abstract

The 26S proteasome is at the executive end of the ubiquitin-proteasome pathway for the controlled degradation of intracellular proteins. While the structure of its 20S core particle (CP) has been determined by X-ray crystallography, the structure of the 19S regulatory particle (RP), which recruits substrates, unfolds them, and translocates them to the CP for degradation, has remained elusive. Here, we describe the molecular architecture of the 26S holocomplex determined by an integrative approach based on data from cryoelectron microscopy, X-ray crystallography, residue-specific chemical cross-linking, and several proteomics techniques. The "lid" of the RP (consisting of Rpn3/5/6/7/8/9/11/12) is organized in a modular fashion. Rpn3/5/6/7/9/12 form a horseshoe-shaped heterohexamer, which connects to the CP and roofs the AAA-ATPase module, positioning the Rpn8/Rpn11 heterodimer close to its mouth. Rpn2 is rigid, supporting the lid, while Rpn1 is conformationally variable, positioned at the periphery of the ATPase ring. The ubiquitin receptors Rpn10 and Rpn13 are located in the distal part of the RP, indicating that they were recruited to the complex late in its evolution. The modular structure of the 26S proteasome provides insights into the sequence of events prior to the degradation of ubiquitylated substrates.

Fig. 1.

Cryo-EM map of the S. pombe 26S proteasome. (A) The single-particle cryo-EM density map of the 26S proteasome from S. pombe at 8.4-Å resolution is shown in two views, related by a 90° rotation around the pseudo-sevenfold axis of the CP (CP: red; AAA-ATPase hexamer: blue; Rpn subunits: gold). (B) The isosurface of the cryo-EM map is colored according to the local resolution in Å, as specified in the color bar.

Fig. 2.

Chemical cross-links for the S. pombe and S. cerevisiae 26S proteasomes. Fifty-five (21) pairs of cross-linked lysines from the S. pombe (S. cerevisiae) 26S proteasome subunits are shown as an interaction network (Table S1). Multiple edges between a pair of subunits indicate multiple cross-linked lysine pairs.

Fig. 3.

Integrative structure determination of the RP. First, structural data and information are generated by various experiments and computational methods, respectively. Second, the AAA-ATPase hexamer and the Rpn subunits are each represented as a string of beads or by atoms, and the data are translated into spatial restraints on their arrangement. Third, an ensemble of structures that satisfy the data are obtained by inferential sampling. Fourth, the ensemble is clustered into distinct subsets of structures, and analyzed in terms of geometry and accuracy.

Fig. 4.

Analysis of the ensemble of 26S proteasome structures. (A) Distribution of scores for the ensemble of models. 5·10⁵ of the nearly 250 million sampled models violated at most five of the 98 cross-linking and proteomics restraints. (B) Clustering of the top scoring models that violate at most five cross-linking and proteomics restraints. The models were clustered by their centroid rmsd into three main clusters with the maximum rmsd from the cluster centroid of 5 Å. (C) Conservation of subunit positions in the ensemble. For each subunit, the average cross-correlation coefficient between pairs of subunit clusters is displayed. The plot shows localization variability for Rpn3, Rpn7, Rpn8, and Rpn11. (D) Localization probabilities for each subunit in the cluster displayed as a heat map. Each voxel of the density map is colored by its localization probability, defined as the percentage of cluster models that localized a subunit in the voxel. A gradient color from blue to red indicates that the subunit was localized to a voxel in 5% to 100% of the models; a gray color indicates 0%.

Fig. 5.

Structure of the AAA-ATPase hexamer. (A) Atomic representation of the CP and AAA-ATPase hexamer after flexible fitting into the cryo-EM map, colored by their C^α atom rmsd compared to the initial comparative models based on the S. cerevisiae CP (13) and the AAA/N-rings from PAN (18). (B) Near the C termini of Rpt2 (left) and Rpt3 (right), we observe density in the pockets formed by the α3/α4 and α1/α2 interfaces, respectively (cyan).

Fig. 6.

Modular architecture of the 26S proteasome. (A) From left to right: segmented densities of the PC-repeat containing proteins, Ub receptors (Rpn13 is taken from the S. cerevisiae 26S proteasome because it binds substoichmetrically in S. pombe) (33), the MPN-domain-containing proteins, and the PCI-domain-containing subunits. (B) The segmented 26S proteasome density in three different views. (C) Hybrid representation of the 26S proteasome, indicating fitted available comparative models for the CP (red), the AAA-ATPase heterohexamer (blue), the PCI heterohexamer (green), Rpn8 MPN domain (pink), Rpn2 LRR domain (yellow), and Rpn10 VWA domain (purple). (D) Close-up view of the heterohexamer formed by the PCI domains.

Fig. 7.

Mechanistic model for the early steps of protein degradation by the 26S proteasome. Polyubiquitylated substrates first bind to the Ub receptors Rpn10 and Rpn13 in concert (33) (I). During the subsequent commitment step (II), the substrates are more tightly bound by the AAA-ATPase coiled coils, which may perform swinging motion. The coiled coils further transfer the substrates to Rpn11, where they are deubiquitylated to enable Ub recycling (III).