The Mechanism of NEDD8 Activation of CUL5 Ubiquitin E3 Ligases

Mol Cell Proteomics. 2021:20:100019. doi: 10.1074/mcp.RA120.002414. Epub 2021 Jan 6.


Cullin RING E3 ligases (CRLs) ubiquitylate hundreds of important cellular substrates. Here we have assembled and purified the Ankyrin repeat and SOCS Box protein 9 CUL5 RBX2 ligase (ASB9-CRL) in vitro and show how it ubiquitylates one of its substrates, CKB. CRLs occasionally collaborate with RING between RING E3 ligases (RBRLs), and indeed, mass spectrometry analysis showed that CKB is specifically ubiquitylated by the ASB9-CRL-ARIH2-UBE2L3 complex. Addition of other E2s such as UBE2R1 or UBE2D2 contributes to polyubiquitylation but does not alter the sites of CKB ubiquitylation. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analysis revealed that CUL5 neddylation allosterically exposes its ARIH2 binding site, promoting high-affinity binding, and it also sequesters the NEDD8 E2 (UBE2F) binding site on RBX2. Once bound, ARIH2 helices near the Ariadne domain active site are exposed, presumably relieving its autoinhibition. These results allow us to propose a model of how neddylation activates ASB-CRLs to ubiquitylate their substrates.

Keywords: hydrogen-deuterium exchange mass spectrometry; post-translational modifications; protein complex; ubiquitin ligase.

Publication types

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

MeSH terms

  • Cullin Proteins / chemistry
  • Cullin Proteins / metabolism*
  • Escherichia coli / genetics
  • NEDD8 Protein / chemistry
  • NEDD8 Protein / metabolism*
  • Suppressor of Cytokine Signaling Proteins / chemistry
  • Suppressor of Cytokine Signaling Proteins / genetics
  • Suppressor of Cytokine Signaling Proteins / metabolism
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination


  • ASB9 protein, human
  • CUL5 protein, human
  • Cullin Proteins
  • NEDD8 Protein
  • NEDD8 protein, human
  • Suppressor of Cytokine Signaling Proteins
  • ARIH2 protein, human
  • RNF7 protein, human
  • Ubiquitin-Protein Ligases