Site-specific inhibition of the small ubiquitin-like modifier (SUMO)-conjugating enzyme Ubc9 selectively impairs SUMO chain formation

J Biol Chem. 2017 Sep 15;292(37):15340-15351. doi: 10.1074/jbc.M117.794255. Epub 2017 Aug 7.


Posttranslational modifications by small ubiquitin-like modifiers (SUMOs) regulate many cellular processes, including genome integrity, gene expression, and ribosome biogenesis. The E2-conjugating enzyme Ubc9 catalyzes the conjugation of SUMOs to ϵ-amino groups of lysine residues in target proteins. Attachment of SUMO moieties to internal lysines in Ubc9 itself can further lead to the formation of polymeric SUMO chains. Mono- and poly-SUMOylations of target proteins provide docking sites for distinct adapter and effector proteins important for regulating discrete SUMO-regulated pathways. However, molecular tools to dissect pathways depending on either mono- or poly-SUMOylation are largely missing. Using a protein-engineering approach, we generated high-affinity SUMO2 variants by phage display that bind the back side binding site of Ubc9 and function as SUMO-based Ubc9 inhibitors (SUBINs). Importantly, we found that distinct SUBINs primarily inhibit poly-SUMO chain formation, whereas mono-SUMOylation was not impaired. Proof-of-principle experiments demonstrated that in a cellular context, SUBINs largely prevent heat shock-triggered poly-SUMOylation. Moreover, SUBINs abrogated arsenic-induced degradation of promyelocytic leukemia protein. We propose that the availability of the new chain-selective SUMO inhibitors reported here will enable a thorough investigation of poly-SUMO-mediated cellular processes, such as DNA damage responses and cell cycle progression.

Keywords: PML; UBC9; enzyme inhibitor; heat shock; nuclear magnetic resonance (NMR); phage display; protein engineering; small ubiquitin-like modifier (SUMO); sumoylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Arsenic / toxicity
  • Binding Sites
  • Binding, Competitive
  • Gene Deletion
  • Gene Library
  • HEK293 Cells
  • HeLa Cells
  • Hot Temperature
  • Humans
  • Ligands
  • Models, Molecular*
  • Peptide Fragments / chemistry
  • Peptide Fragments / genetics
  • Peptide Fragments / metabolism
  • Point Mutation
  • Promyelocytic Leukemia Protein / antagonists & inhibitors
  • Promyelocytic Leukemia Protein / chemistry
  • Promyelocytic Leukemia Protein / genetics
  • Promyelocytic Leukemia Protein / metabolism*
  • Protein Interaction Domains and Motifs
  • RNA Interference
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Small Ubiquitin-Related Modifier Proteins / chemistry
  • Small Ubiquitin-Related Modifier Proteins / genetics
  • Small Ubiquitin-Related Modifier Proteins / metabolism*
  • Sumoylation* / drug effects
  • Ubiquitin-Conjugating Enzymes / antagonists & inhibitors
  • Ubiquitin-Conjugating Enzymes / chemistry
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / metabolism*


  • Ligands
  • Peptide Fragments
  • Promyelocytic Leukemia Protein
  • Recombinant Fusion Proteins
  • Recombinant Proteins
  • SUMO2 protein, human
  • Small Ubiquitin-Related Modifier Proteins
  • PML protein, human
  • Ubiquitin-Conjugating Enzymes
  • ubiquitin-conjugating enzyme UBC9
  • Arsenic

Associated data

  • PDB/2ckh
  • PDB/5d2m