Structure and energetics of pairwise interactions between proteasome subunits RPN2, RPN13, and ubiquitin clarify a substrate recruitment mechanism

Abstract

The 26S proteasome is a large cellular assembly that mediates the selective degradation of proteins in the nucleus and cytosol and is an established target for anticancer therapeutics. Protein substrates are typically targeted to the proteasome through modification with a polyubiquitin chain, which can be recognized by several proteasome-associated ubiquitin receptors. One of these receptors, RPN13/ADRM1, is recruited to the proteasome through direct interaction with the large scaffolding protein RPN2 within the 19S regulatory particle. To better understand the interactions between RPN13, RPN2, and ubiquitin, we used human proteins to map the RPN13-binding epitope to the C-terminal 14 residues of RPN2, which, like ubiquitin, binds the N-terminal pleckstrin-like receptor of ubiquitin (PRU) domain of RPN13. We also report the crystal structures of the RPN13 PRU domain in complex with peptides corresponding to the RPN2 C terminus and ubiquitin. Through mutational analysis, we validated the RPN2-binding interface revealed by our structures and quantified binding interactions with surface plasmon resonance and fluorescence polarization. In contrast to a previous report, we find that RPN13 binds ubiquitin with an affinity similar to that of other proteasome-associated ubiquitin receptors and that RPN2, ubiquitin, and the deubiquitylase UCH37 bind to RPN13 with independent energetics. These findings provide a detailed characterization of interactions that are important for proteasome function, indicate ubiquitin affinities that are consistent with the role of RPN13 as a proteasomal ubiquitin receptor, and have major implications for the development of novel anticancer therapeutics.

Structural basis for the recognition of K48-linked Ub chain by proteasomal receptor Rpn13

“…We found that, titrating unlabeled K48-diUb to 15 N-labeled Rpn13 causes large chemical shift perturbations (CSPs), which mainly involve residues 73-83 and 93-106 ( Fig 3a). These residues form a contiguous surface on Rpn13, covering an area larger than expected from the previously determined complex structure between Rpn13 NTD and Ub monomer 6,7 . On the other hand, titrating unlabeled Rpn13 NTD to K48-dUb, with either proximal or distal Ub 15 N-labeled and the other subunit unlabeled, the CSPs are mainly observed for residues in the β-sheet region of both Ub subunits.…”

“…This would explain a 2:1 stoichiometry between K48-diUb and Rpn13 NTD , or a 1:1 stoichiometry between Ub monomer and Rnpn13 NTD previously observed in bulk titrations 3,7,9 . Indeed, the Rpn13 NTD -Ub complex has been crystalized as a dimer ( Supplementary Fig. S14) 7 . Herein, the structure of Rpn13 NTD :K48-diUb complex clearly explains how the two Ub subunits of K48-diUb simultaneously interact with Rpn13 NTD .…”

“…First, biochemical and mass spectrometry analyses showed that only one copy of Rpn13 exists in the 26S proteasome, which means that Rpn13 is at 1:2 molar ratio with other regulatory proteins in the 19S particles 12 . Second, Rpn13 is recruited to the proteasome by interacting with the Cterminal tail of Rpn2 7,13 , which is largely unstructured 11,14 . Therefore, Rpn13 is mobile with respect to the proteasome.…”

Proteasome‐mediated proteostasis: Novel medicinal and pharmacological strategies for diseases

“…In another observation, different binding interactions between proteasome subunits, viz. RPN13, RPN2, and ubiquitin, were substantiated for substrate recruitment in proteasome, using surface plasmon resonance and fluorescence polarization . The gating mechanism and the energetics of 20S proteasome core particle in T. acidophilum were shown by solution NMR technique …”

“…RPN13, RPN2, and ubiquitin, were substantiated for substrate recruitment in proteasome, using surface plasmon resonance and fluorescence polarization. 102 The gating mechanism and the energetics of 20S proteasome core particle in T. acidophilum were shown by solution NMR technique. 103 Ubiquitous involvement of proteasome in different cellular pathways and the wider linkages due to lack of functions associated with many diseases has prompted scientists to propose this multiproteinase complex as a suitable target for remedial interventions.…”