E3 ligase autoinhibition by C-degron mimicry maintains C-degron substrate fidelity

Mol Cell. 2023 Mar 2;83(5):770-786.e9. doi: 10.1016/j.molcel.2023.01.019. Epub 2023 Feb 16.


E3 ligase recruitment of proteins containing terminal destabilizing motifs (degrons) is emerging as a major form of regulation. How those E3s discriminate bona fide substrates from other proteins with terminal degron-like sequences remains unclear. Here, we report that human KLHDC2, a CRL2 substrate receptor targeting C-terminal Gly-Gly degrons, is regulated through interconversion between two assemblies. In the self-inactivated homotetramer, KLHDC2's C-terminal Gly-Ser motif mimics a degron and engages the substrate-binding domain of another protomer. True substrates capture the monomeric CRL2KLHDC2, driving E3 activation by neddylation and subsequent substrate ubiquitylation. Non-substrates such as NEDD8 bind KLHDC2 with high affinity, but its slow on rate prevents productive association with CRL2KLHDC2. Without substrate, neddylated CRL2KLHDC2 assemblies are deactivated via distinct mechanisms: the monomer by deneddylation and the tetramer by auto-ubiquitylation. Thus, substrate specificity is amplified by KLHDC2 self-assembly acting like a molecular timer, where only bona fide substrates may bind before E3 ligase inactivation.

Keywords: Autoinhibition; C-END degron; CUL2; Cullin-RING Ligase; E3; KLHDC10; KLHDC3; Kinetic proofreading; NEDD8; Targeted protein degradation; allostery; higher-order assembly; protein-protein interaction; ubiquitin.

Publication types

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

MeSH terms

  • Carrier Proteins
  • Humans
  • Proteins* / metabolism
  • Ubiquitin-Protein Ligases* / metabolism


  • Carrier Proteins
  • Proteins
  • Ubiquitin-Protein Ligases
  • KLHDC2 protein, human