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.
Highlights
Rpn9 is a subunit of the proteasome regulatory particle
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 proteasome-COP9/CSN-initiation factor (PCI)-PCI interactions in the lid
Characterizations of Rpn9 Architecture—The S. cerevisiae Rpn9 is a 393-residue protein with a molecular mass of 45 kDa
Summary
Rpn is a subunit of the proteasome regulatory particle. Results: Rpn interacts with Rpn and Rpn via its N-terminal ␣-solenoid and C-terminal proteasome-COP9/CSN-initiation factor (PCI) domains, respectively. Based on sequence homology and domain architecture, the lid subunits can be classified into two groups; Rpn and Rpn both contain an Mpr1/Pad N-terminal (MPN) domain (19 –22), whereas the other six subunits all share a C-terminal proteasome-COP9/ CSN-initiation factor (PCI) domain. The PCI domain is commonly found in three important multiprotein complexes in cells, namely the proteasome, the COP9/CSN signalosome, and the initiation factor eIF3, and is proposed to have essential roles in subunit interactions and complex assembly [23]. Rpn is a PCI domain-containing lid subunit and is necessary for the integrity and efficiency of the 26 S proteasome [29, 30] It interacts with the ubiquitin receptor Rpn, and the ⌬rpn Saccharomyces cerevisiae strain was reported to accumulate multiubiquitinated proteins at restrictive temperatures [29]. The results shed new light on the regulation of PCI-mediated lid assembly
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