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

Protein ubiquitination is a common form of post-translational modification that regulates a broad spectrum of protein substrates in diverse cellular pathways(1). 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(2,3). Conserved from yeast to human, the DDB1 - CUL4 - ROC1 complex is a recently identified cullin-RING ubiquitin ligase, which regulates DNA repair(4-10), DNA replication(11-14) and transcription(15), and can also be subverted by pathogenic viruses to benefit viral infection(16). 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.