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. 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.
Angers
Stephane
S
Howard Hughes Medical Institute, University of Washington, School of Medicine, Box 357280, Seattle, Washington 98195, USA.
Li
Ti
T
Yi
Xianhua
X
MacCoss
Michael J
MJ
Moon
Randall T
RT
Zheng
Ning
N
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D044842:Cullin Proteins / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018583:Pliability; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D040901:Proteomics; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D025801:Ubiquitin / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism*
C114234:CUL4A protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; C091435:DDB1 protein, human; D004268:DNA-Binding Proteins; C479529:RBX1 protein, human; D025801:Ubiquitin; D044767:Ubiquitin-Protein Ligases