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

• Published in: Nature 2006-10, Vol.443 (7111), p.590-593
• Main Authors: Moon, Randall T, Zheng, Ning, Yi, Xianhua, Angers, Stephane, MacCoss, Michael J, Li, Ti
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Binding Sites; Biological and medical sciences; Carrier Proteins - chemistry; Carrier Proteins - metabolism; Cellular biology; Crystallography, X-Ray; Cullin Proteins - chemistry; Cullin Proteins - metabolism; Deoxyribonucleic acid; DNA; DNA repair; DNA-Binding Proteins - chemistry; DNA-Binding Proteins - metabolism; Enzymes; Fundamental and applied biological sciences. Psychology; Humanities and Social Sciences; Humans; Interactions. Associations; Intermolecular phenomena; letter; Mass spectrometry; Models, Molecular; Molecular biophysics; Molecular Sequence Data; Molecular structure; multidisciplinary; Pliability; Protein Binding; Protein Structure, Quaternary; Proteins; Proteomics; Science; Science (multidisciplinary); Structure-Activity Relationship; Substrate Specificity; Substrates; Ubiquitin - metabolism; Ubiquitin-Protein Ligases - chemistry; Ubiquitin-Protein Ligases - metabolism; Viruses; Yeasts
• ISSN: 0028-0836; 1476-4687; 1476-4679
• DOI: 10.1038/nature05175

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 β-propeller domain for cullin scaffold binding and a variably attached separate double-β-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.