Molecular architecture and assembly of the DDB1-CUL4A ubiquitin ligase machinery

Nature. 2006 Oct 5;443(7111):590-3. doi: 10.1038/nature05175.

Abstract

Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways. Through a three-enzyme (E1-E2-E3) cascade, the attachment of ubiquitin to proteins is catalysed by the E3 ubiquitin ligase, which is best represented by the superfamily of the cullin-RING complexes. Conserved from yeast to human, the DDB1-CUL4-ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair, DNA replication and transcription, and can also be subverted by pathogenic viruses to benefit viral infection. Lacking a canonical SKP1-like cullin adaptor and a defined substrate recruitment module, how the DDB1-CUL4-ROC1 E3 apparatus is assembled for ubiquitinating various substrates remains unclear. Here we present crystallographic analyses of the virally hijacked form of the human DDB1-CUL4A-ROC1 machinery, which show that DDB1 uses one beta-propeller domain for cullin scaffold binding and a variably attached separate double-beta-propeller fold for substrate presentation. Through tandem-affinity purification of human DDB1 and CUL4A complexes followed by mass spectrometry analysis, we then identify a novel family of WD40-repeat proteins, which directly bind to the double-propeller fold of DDB1 and serve as the substrate-recruiting module of the E3. Together, our structural and proteomic results reveal the structural mechanisms and molecular logic underlying the assembly and versatility of a new family of cullin-RING E3 complexes.

Publication types

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

MeSH terms

  • Amino Acid Motifs
  • Amino Acid Sequence
  • Binding Sites
  • Carrier Proteins / chemistry
  • Carrier Proteins / metabolism
  • Crystallography, X-Ray
  • Cullin Proteins / chemistry*
  • Cullin Proteins / metabolism*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / metabolism*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Pliability
  • Protein Binding
  • Protein Structure, Quaternary
  • Proteomics
  • Structure-Activity Relationship
  • Substrate Specificity
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / chemistry*
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • CUL4A protein, human
  • Carrier Proteins
  • Cullin Proteins
  • DDB1 protein, human
  • DNA-Binding Proteins
  • RBX1 protein, human
  • Ubiquitin
  • Ubiquitin-Protein Ligases