Solution structure of yeast Rpn9: insights into proteasome lid assembly

Abstract

The regulatory particle (RP) of the 26 S proteasome functions in preparing polyubiquitinated substrates for degradation. The lid complex of the RP contains an Rpn8-Rpn11 heterodimer surrounded by a horseshoe-shaped scaffold formed by six proteasome-COP9/CSN-initiation factor (PCI)-containing subunits. The PCI domains are essential for lid assembly, whereas the detailed molecular mechanisms remain elusive. Recent cryo-EM studies at near-atomic resolution provided invaluable information on the RP architecture in different functional states. Nevertheless, atomic resolution structural information on the RP is still limited, and deeper understanding of RP assembly mechanism requires further studies on the structures and interactions of individual subunits or subcomplexes. Herein we report the high-resolution NMR structures of the PCI-containing subunit Rpn9 from Saccharomyces cerevisiae. The 45-kDa protein contains an all-helical N-terminal domain and a C-terminal PCI domain linked via a semiflexible hinge. The N-terminal domain mediates interaction with the ubiquitin receptor Rpn10, whereas the PCI domain mediates interaction with the neighboring PCI subunit Rpn5. The Rpn9-Rpn5 interface highlights two structural motifs on the winged helix module forming a hydrophobic center surrounded by ionic pairs, which is a common pattern for all PCI-PCI interactions in the lid. The results suggest that divergence in surface composition among different PCI pairs may contribute to the modulation of lid assembly.

Keywords: Nuclear Magnetic Resonance (NMR); PCI; Peptide Interaction; Proteasome; Protein Assembly; Protein Structure; Regulatory Particle; Rpn9.

FIGURE 1.

Limited proteolysis of S. cerevisiae Rpn9. A, SDS-PAGE showing the results of limited trypsin digestion of yeast Rpn9. The digestion reaction was performed at room temperature with an Rpn9/trypsin molar ratio of 100:1. B, Rpn9 architecture based on limited proteolysis and constructs used in this study. The cleavage sites are marked with red arrows, and the corresponding residues are labeled.

FIGURE 2.

Solution structures of the NTD and PCI domains of S. cerevisiae Rpn9. A and B, ribbon diagrams and 20 lowest energy structure ensembles of the NTD (A) and PCI (B) domains of yeast Rpn9. Secondary structural elements are labeled and numbered according to the full-length structure.

FIGURE 3.

Comparison of the Rpn9-PCI structure with other PCI domains. Shown are ribbon diagrams of S. cerevisiae Rpn9 PCI domain (A), A. thaliana CSN7 PCI domain (B; PDB code 3CHM), H. sapiens eIF3K subunit (C; PDB code 1RZ4), S. pombe Rpn12 subunit (D; PDB code 4B0Z) and D. melanogaster Rpn6 subunit (E; PDB code 3TXN). The central helices are shown in gray.

FIGURE 4.

Solution structures of S. cerevisiae Rpn9. A, ribbon diagrams and 20 lowest energy structure ensembles of the yeast Rpn9. The NTD, hinge, and PCI regions are colored as in Fig. 1. Secondary structures of the hinge region are labeled. B, local structure of the hinge region showing the residues that have the largest contribution to the observed interhelical NOEs. C, ribbon diagram of Rpn9 showing the five pairs of anti-parallel double helices. The first and second helices in each repeat are colored red and blue, respectively. The helix α1, which is not part of the right-handed superhelical solenoid, is colored yellow. The molecule is rotated compared with A to obtain a better view of the double-helical repeats. D, ensembles of 20 lowest energy structures of Rpn9 aligned using the NTD (left) and the PCI (right) domains, respectively.

FIGURE 5.

Fitting of S. cerevisiae Rpn9 structure into EM density. A, location of Rpn9 in the 7.4 Å cryo-EM density of the S. cerevisiae 26 S proteasome (EMDB code 2165). The 20 S CP is clipped off, and the 19 S RP subunits are colored and labeled. The horseshoe-shaped structure formed by six PCI-containing subunits, Rpn9-Rpn5-Rpn6-Rpn7-Rpn3-Rpn12, is shown on the right. B, detailed view of the Rpn9 fitted into the EM envelope. Density assigned for Rpn9 was segmented from the map. C, comparison of the Rpn9 structure fitted to the EM density (green) with two models from the 20-conformer NMR structure ensemble. The conformer showing the lowest backbone r.m.s. deviation (3.9 Å) with the fitted structure is shown in blue, and the other showing the highest backbone r.m.s. deviation (7.7 Å) with the fitted structure is shown in red. D, surface conservation of Rpn9 with a cyan-white-magenta color gradient representing increasing conservation. The conservation score is calculated using the multiple-sequence alignment of 21 representative sequences from S. cerevisiae, Neurospora crassa, Talaromyces stipitatus, Yarrowia lipolytica, S. pombe, Candida albicans, H. sapiens, Bos taurus, Mus musculus, Rattus norvegicus, Gallus gallus, D. melanogaster, Caenorhabditis elegans, A. thaliana, Physcomitrella patens, Volvox carteri, Naegleria gruberi, Dictyostelium discoideum, Paramecium tetraurelia, Trichomonas vaginalis, and Trypanosoma brucei. The structure of Rpn9 fitted to the EM map is used, and three highly conserved regions are indicated.

FIGURE 6.

Interaction between Rpn9 and Rpn10 subunits. A, peak intensity (top) and chemical shift (bottom) changes of [¹⁵N]Rpn9-NTD when titrated with unlabeled Rpn10. The intensities were taken from spectra of free [¹⁵N]Rpn9-NTD and a sample with a [¹⁵N]Rpn9-NTD/Rpn10 molar ratio of 1:2. The intensity ratios were calculated and normalized using a number of peaks least affected. The composite chemical shift changes were calculated using the empirical equation, Δδ_comp = √(Δδ_H² + (Δδ_N²/6)²). B, mapping of NMR titration results onto the Rpn9 structure. Residues showing significant peak intensity changes (normalized intensity ratio <0.5) are colored red. Residues with chemical shift perturbations (composite chemical shift changes >0.02) are shown in stick representations and colored red. C, local structure of the Rpn9-Rpn10 interface based on fitting of the Rpn9 NMR structure into the EM density. Important residues contributing to the contact surface are shown in stick representations on the right. D, multiple-sequence alignment of the Rpn9 and Rpn10 subunits showing the segments locating on the interface. E, normalized peak intensity changes of [¹⁵N]Rpn9-NTD K39E/F39A mutant when titrated with unlabeled Rpn10 at a molar ratio of 1:2. Error bars, S.E.

FIGURE 7.

Interaction between Rpn9 and Rpn5 subunits. A, size exclusion chromatography of Rpn9·Rpn5 heterodimeric complex compared with Rpn9 and Rpn5 proteins alone. B, an overlay of the ¹H-¹⁵N HSQC spectra of the [¹⁵N]Rpn9-PCI domain and the [¹⁵N]Rpn9-PCI·Rpn5 complex purified by size exclusion chromatography. Changes of chemical shifts are shown at the bottom. Gray bars indicate the residues that disappeared or show chemical shift changes too large to be traced. C, local structure of the Rpn9-Rpn5 interface. Amino acids showing significant chemical shift changes (composite chemical shift changes larger than 0.1 and those that could not be traced) are colored blue on the left. Important residues contributing to the contact surface are shown in stick representations on the right. D, multiple-sequence alignment of the Rpn9 and Rpn5 subunits showing only the segments located on the interface. E, size exclusion chromatography of Rpn9-Rpn5 interactions using mutant proteins. Dashed lines, expected positions corresponding to Rpn9·Rpn5 heterodimer (black), Rpn5 (red), and Rpn9 (blue), respectively, as shown in A.

FIGURE 8.

PCI-PCI interaction in the proteasome lid via the WH module. Local structures and multiple-sequence alignments of PCI pairs Rpn5-Rpn6, Rpn6-Rpn7, Rpn7-Rpn3, and Rpn3-Rpn12 are shown in A–D. The atomic coordinates are from the EM density-based RP model (PDB code 4B4T). Residues that possibly contribute to the interaction are shown in stick representations, and the strictly conserved residues are labeled in boldface italic type.

FIGURE 9.

Common motifs for PCI interactions in proteasome RP. The two structural motifs mediating PCI-PCI are indicated by a red rectangle and blue ellipse. Motif A in the Rpn9 subunit and motif B in the Rpn12 subunit are less conserved and are shown by a gray ellipse and rectangle.