Structures of Rpn1 T1:Rad23 and hRpn13:hPLIC2 Reveal Distinct Binding Mechanisms between Substrate Receptors and Shuttle Factors of the Proteasome

Structure. 2016 Aug 2;24(8):1257-1270. doi: 10.1016/j.str.2016.05.018. Epub 2016 Jul 7.

Abstract

Three receptors (Rpn1/S2/PSMD2, Rpn10/S5a, Rpn13/Adrm1) in the proteasome bind substrates by interacting with conjugated ubiquitin chains and/or shuttle factors (Rad23/HR23, Dsk2/PLIC/ubiquilin, Ddi1) that carry ubiquitinated substrates to proteasomes. We solved the structure of two such receptors with their preferred shuttle factor, namely hRpn13(Pru):hPLIC2(UBL) and scRpn1 T1:scRad23(UBL). We find that ubiquitin folds in Rad23 and Dsk2 are fine-tuned by residue substitutions to achieve high affinity for Rpn1 and Rpn13, respectively. A single substitution in hPLIC2 yields enhanced interactions with the Rpn13 ubiquitin contact surface and sterically blocks hRpn13 binding to its preferred ubiquitin chain type, K48-linked chains. Rpn1 T1 binds two ubiquitins in tandem and we find that Rad23 binds exclusively to the higher-affinity Helix28/Helix30 site. Rad23 contacts at Helix28/Helix30 are optimized compared to ubiquitin by multiple conservative amino acid substitutions. Thus, shuttle factors deliver substrates to proteasomes through fine-tuned ubiquitin-like surfaces.

Keywords: Dsk2; Rad23; Rpn1; Rpn13; proteasome; ubiquitin.

MeSH terms

  • Adaptor Proteins, Signal Transducing
  • Amino Acid Sequence
  • Amino Acid Substitution
  • Autophagy-Related Proteins
  • Binding Sites
  • Cell Cycle Proteins / chemistry*
  • Cell Cycle Proteins / genetics
  • Cell Cycle Proteins / metabolism
  • Crystallography, X-Ray
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism
  • Gene Expression
  • Humans
  • Intracellular Signaling Peptides and Proteins
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / genetics
  • Membrane Glycoproteins / metabolism
  • Molecular Docking Simulation
  • Molecular Dynamics Simulation
  • Mutation
  • 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
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Saccharomyces cerevisiae / chemistry*
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry*
  • Saccharomyces cerevisiae Proteins / genetics
  • Saccharomyces cerevisiae Proteins / metabolism
  • Sequence Alignment
  • Sequence Homology, Amino Acid
  • Substrate Specificity
  • Thermodynamics
  • Ubiquitins / chemistry*
  • Ubiquitins / genetics
  • Ubiquitins / metabolism

Substances

  • ADRM1 protein, human
  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Proteins
  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Intracellular Signaling Peptides and Proteins
  • Membrane Glycoproteins
  • RAD23 protein, S cerevisiae
  • RPN1 protein, S cerevisiae
  • Recombinant Proteins
  • Saccharomyces cerevisiae Proteins
  • UBQLN2 protein, human
  • Ubiquitins
  • Proteasome Endopeptidase Complex