Role of E2-RING Interactions in Governing RNF4-Mediated Substrate Ubiquitination

J Mol Biol. 2016 Nov 20;428(23):4639-4650. doi: 10.1016/j.jmb.2016.09.018. Epub 2016 Sep 24.


Members of the really interesting new gene (RING) E3 ubiquitin ligase family bind to both substrate and ubiquitin-charged E2 enzyme, promoting the transfer of ubiquitin from the E2 to substrate. Either a single ubiquitin or one of the several types of polyubiquitin chains can be conjugated to substrate proteins, with different types of ubiquitin modifications signaling the distinct outcomes. E2 enzymes play a central role in governing the nature of the ubiquitin modification, although the essential features of the E2 that differentiate mono- versus polyubiquitinating E2 enzymes remain unclear. RNF4 is a compact RING E3 ligase that directs the ubiquitination of polySUMO chains in concert with several different E2 enzymes. RNF4 monoubiquitinates polySUMO substrates in concert with RAD6B and polyubiquitinates substrates together with UBCH5B, a promiscuous E2 that can function with a broad range of E3 ligases. We find that the divergent ubiquitination activities of RAD6B and UBCH5B are governed by differences at the RING-binding surface of the E2. By mutating the RAD6B RING-binding surface to resemble that of UBCH5B, RAD6B can be transformed into a highly active UBCH5B-like E2 that polyubiquitinates SUMO chains in concert with RNF4. The switch in RAD6B activity correlates with increased affinity of the E2 for RNF4. These results point to an important role of the affinity between an E3 and its cognate E2 in governing the multiplicity of substrate ubiquitination.

Keywords: E2 ubiquitin-conjugating enzyme; E3 ubiquitin ligase; RAD6B; RING finger protein 4 (RNF4); ubiquitin.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Humans
  • Nuclear Proteins / metabolism*
  • Protein Processing, Post-Translational*
  • Small Ubiquitin-Related Modifier Proteins / metabolism*
  • Transcription Factors / metabolism*
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitination*


  • Nuclear Proteins
  • RNF4 protein, human
  • Small Ubiquitin-Related Modifier Proteins
  • Transcription Factors
  • UBE2B protein, human
  • UBE2D2 protein, human
  • Ubiquitin-Conjugating Enzymes