Structural basis of Cullin 2 RING E3 ligase regulation by the COP9 signalosome

Nat Commun. 2019 Aug 23;10(1):3814. doi: 10.1038/s41467-019-11772-y.

Abstract

Cullin-Ring E3 Ligases (CRLs) regulate a multitude of cellular pathways through specific substrate receptors. The COP9 signalosome (CSN) deactivates CRLs by removing NEDD8 from activated Cullins. Here we present structures of the neddylated and deneddylated CSN-CRL2 complexes by combining single-particle cryo-electron microscopy (cryo-EM) with chemical cross-linking mass spectrometry (XL-MS). These structures suggest a conserved mechanism of CSN activation, consisting of conformational clamping of the CRL2 substrate by CSN2/CSN4, release of the catalytic CSN5/CSN6 heterodimer and finally activation of the CSN5 deneddylation machinery. Using hydrogen-deuterium exchange (HDX)-MS we show that CRL2 activates CSN5/CSN6 in a neddylation-independent manner. The presence of NEDD8 is required to activate the CSN5 active site. Overall, by synergising cryo-EM with MS, we identify sensory regions of the CSN that mediate its stepwise activation and provide a framework for understanding the regulatory mechanism of other Cullin family members.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / isolation & purification
  • Adaptor Proteins, Signal Transducing / metabolism
  • Animals
  • COP9 Signalosome Complex / isolation & purification
  • COP9 Signalosome Complex / metabolism
  • COP9 Signalosome Complex / ultrastructure*
  • Cryoelectron Microscopy
  • Intracellular Signaling Peptides and Proteins / isolation & purification
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Mass Spectrometry
  • NEDD8 Protein / isolation & purification
  • NEDD8 Protein / metabolism
  • NEDD8 Protein / ultrastructure*
  • Peptide Hydrolases / isolation & purification
  • Peptide Hydrolases / metabolism
  • Peptide Hydrolases / ultrastructure*
  • Protein Processing, Post-Translational
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Recombinant Proteins / ultrastructure
  • Sf9 Cells
  • Ubiquitin-Protein Ligases / isolation & purification
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitin-Protein Ligases / ultrastructure*

Substances

  • Adaptor Proteins, Signal Transducing
  • COPS6 protein, human
  • Intracellular Signaling Peptides and Proteins
  • NEDD8 Protein
  • NEDD8 protein, human
  • Recombinant Proteins
  • CULL-RING ligase, human
  • Ubiquitin-Protein Ligases
  • Peptide Hydrolases
  • COPS5 protein, human
  • COP9 Signalosome Complex