Cryo-EM Reveals Unanchored M1-Ubiquitin Chain Binding at hRpn11 of the 26S Proteasome

Structure. 2020 Nov 3;28(11):1206-1217.e4. doi: 10.1016/j.str.2020.07.011. Epub 2020 Aug 11.

Abstract

The 26S proteasome is specialized for regulated protein degradation and formed by a dynamic regulatory particle (RP) that caps a hollow cylindrical core particle (CP) where substrates are proteolyzed. Its diverse substrates unify as proteasome targets by ubiquitination. We used cryogenic electron microscopy (cryo-EM) to study how human 26S proteasome interacts with M1-linked hexaubiquitin (M1-Ub6) unanchored to a substrate and E3 ubiquitin ligase E6AP/UBE3A. Proteasome structures are available with model substrates extending through the RP ATPase ring and substrate-conjugated K63-linked ubiquitin chains present at inhibited deubiquitinating enzyme hRpn11 and the nearby ATPase hRpt4/hRpt5 coiled coil. In this study, we find M1-Ub6 at the hRpn11 site despite the absence of conjugated substrate, indicating that ubiquitin binding at this location does not require substrate interaction with the RP. Moreover, unanchored M1-Ub6 binds to this hRpn11 site of the proteasome with the CP gating residues in both the closed and opened conformational states.

Keywords: E6AP; Rpn11; UBE3A; cryo-EM; deubiquitinating enzyme; linear ubiquitin chains; proteasome; protein degradation; ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Adenosine Triphosphatases / chemistry*
  • Adenosine Triphosphatases / genetics
  • Adenosine Triphosphatases / metabolism
  • Binding Sites
  • Cryoelectron Microscopy
  • Gene Expression
  • Humans
  • Molecular Docking Simulation
  • Polyubiquitin / chemistry*
  • Polyubiquitin / genetics
  • Polyubiquitin / metabolism
  • Proteasome Endopeptidase Complex / chemistry*
  • Proteasome Endopeptidase Complex / genetics
  • Proteasome Endopeptidase Complex / metabolism
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Proteolysis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Trans-Activators / chemistry*
  • Trans-Activators / genetics
  • Trans-Activators / metabolism
  • Ubiquitin / chemistry*
  • Ubiquitin / genetics
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / chemistry*
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination

Substances

  • PSMD14 protein, human
  • Recombinant Proteins
  • Trans-Activators
  • Ubiquitin
  • Polyubiquitin
  • UBE3A protein, human
  • Ubiquitin-Protein Ligases
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease
  • Adenosine Triphosphatases