ABSTRACT
The Elongin complex was originally identified as an RNA polymerase II (RNAPII) elongation factor and subsequently as the substrate recognition component of a Cullin-RING E3 ubiquitin ligase. More recent evidence indicates that the Elongin ubiquitin ligase assembles with the Cockayne syndrome B helicase (CSB) in response to DNA damage and can target stalled polymerases for ubiquitylation and removal from the genome. In this report, we present evidence that the CSB-Elongin ubiquitin ligase pathway has roles beyond the DNA damage response in the activation of RNAPII-mediated transcription. We observed that assembly of the CSB-Elongin ubiquitin ligase is induced not just by DNA damage, but also by a variety of signals that activate RNAPII-mediated transcription, including endoplasmic reticulum (ER) stress, amino acid starvation, retinoic acid, glucocorticoids, and doxycycline treatment of cells carrying several copies of a doxycycline-inducible reporter. Using glucocorticoid receptor (GR)-regulated genes as a model, we showed that glucocorticoid-induced transcription is accompanied by rapid recruitment of CSB and the Elongin ubiquitin ligase to target genes in a step that depends upon the presence of transcribing RNAPII on those genes. Consistent with the idea that the CSB-Elongin pathway plays a direct role in GR-regulated transcription, mouse cells lacking the Elongin subunit Elongin A exhibit delays in both RNAPII accumulation on and dismissal from target genes following glucocorticoid addition and withdrawal, respectively. Taken together, our findings bring to light a new role for the CSB-Elongin pathway in RNAPII-mediated transcription.
Subject(s)
DNA Helicases/genetics , DNA Repair Enzymes/genetics , Elongin/genetics , Poly-ADP-Ribose Binding Proteins/genetics , RNA Polymerase II/genetics , Ubiquitin-Protein Ligases/genetics , Animals , Cockayne Syndrome/enzymology , Cockayne Syndrome/genetics , DNA Helicases/chemistry , DNA Helicases/ultrastructure , DNA Repair/genetics , DNA Repair Enzymes/chemistry , DNA Repair Enzymes/ultrastructure , Elongin/chemistry , Elongin/ultrastructure , Humans , Mice , Multiprotein Complexes/chemistry , Multiprotein Complexes/genetics , Multiprotein Complexes/ultrastructure , Poly-ADP-Ribose Binding Proteins/chemistry , Poly-ADP-Ribose Binding Proteins/ultrastructure , RNA Polymerase II/chemistry , Receptors, Glucocorticoid/chemistry , Receptors, Glucocorticoid/genetics , Ubiquitin/chemistry , Ubiquitin/genetics , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/ultrastructure , Ubiquitination/geneticsABSTRACT
Human immunodeficiency virus-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 (A3) antiviral proteins. Vif folds when it binds Cullin-RING E3 ligase 5 and the transcription cofactor CBF-ß. A five-protein complex containing the substrate receptor (Vif, CBF-ß, Elongin-B, Elongin-C (VCBC)) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use molecular dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and an A3 protein bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, whereas VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation-optimized spectroscopy NMR data, which indicates that the VCBC complex without CUL5 is dynamic on the µs-ms timescale. Our NMR data also show that the VCBC complex is more conformationally restricted when bound to the antiviral APOBEC3F (one of the A3 proteins), consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjunction with the global dynamics of the complex. Like other substrate receptors, VCBC can exist alone or in complex with CUL5 and other proteins in cells. Accordingly, the VCBC complex could be a good target for therapeutics that would inhibit full assembly of the ubiquitination complex by stabilizing an alternate VCBC conformation.
Subject(s)
Cullin Proteins/chemistry , Cytidine Deaminase/chemistry , Molecular Dynamics Simulation , vif Gene Products, Human Immunodeficiency Virus/chemistry , APOBEC Deaminases , Core Binding Factor beta Subunit/chemistry , Crystallization , Elongin/chemistry , Humans , Kinetics , Protein Binding , Protein Conformation , Protein Folding , Structure-Activity Relationship , UbiquitinationABSTRACT
Nucleotide excision repair (NER) is a versatile system that deals with various bulky and helix-distorting DNA lesions caused by UV and environmental mutagens. Based on how lesion recognition occurs, NER has been separated into global genome repair (GGR) and transcription-coupled repair (TCR). The yeast Rad7-Rad16 complex is indispensable for the GGR sub-pathway. Rad7-Rad16 binds to UV-damaged DNA in a synergistic fashion with Rad4, the main lesion recognizer, to achieve efficient recognition of lesions. In addition, Rad7-Rad16 associates with Elc1 and Cul3 to form an EloC-Cul-SOCS-box (ECS)-type E3 ubiquitin ligase complex that ubiquitinates Rad4 in response to UV radiation. However, the structure and architecture of the Rad7-Rad16-Elc1-Cul3 complex remain unsolved. Here, we determined the structure of the Rad7-Elc1 complex and revealed key interaction regions responsible for the formation of the Rad7-Rad16-Elc1-Cul3 complex. These results provide new insights into the assembly of the Rad7-Rad16-Elc1-Cul3 complex and structural framework for further studies.
Subject(s)
Adenosine Triphosphatases/metabolism , Cullin Proteins/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Elongin/chemistry , Elongin/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Crystallography, X-Ray , Models, Molecular , Protein Multimerization , Protein Structure, QuaternaryABSTRACT
Beyond the targeting of E3 ubiquitin ligases to inhibit protein homeostasis, E3 ligase binders can be repurposed as targeted protein degraders (PROTACs or molecular glues). We sought to identify new binders of the VHL E3 ligase by biophysical fragment-based screening followed by X-ray crystallographic soaking. We identified fragments binding at the ElonginC:Cullin2 interface and a new cryptic pocket in VHL, along with other potential ligandable sites predicted computationally and found to bind solvent molecules in crystal structures. The elucidated interactions provide starting points for future ligand development.
Subject(s)
Drug Evaluation, Preclinical/methods , Multiprotein Complexes/chemistry , Ubiquitin-Protein Ligases/metabolism , Von Hippel-Lindau Tumor Suppressor Protein/metabolism , Binding Sites , Crystallography, X-Ray , Elongin/chemistry , Elongin/metabolism , Fluorometry/methods , Humans , Ligands , Magnetic Resonance Spectroscopy , Multiprotein Complexes/metabolism , Polycythemia/genetics , Small Molecule Libraries/chemistry , Small Molecule Libraries/pharmacology , Ubiquitin-Protein Ligases/chemistry , Von Hippel-Lindau Tumor Suppressor Protein/chemistryABSTRACT
Cullin RING E3 ubiquitin ligases (CRLs) function in the ubiquitin proteasome system to catalyze the transfer of ubiquitin from E2 conjugating enzymes to specific substrate proteins. CRLs are large dynamic complexes and attractive drug targets for the development of small-molecule inhibitors and chemical inducers of protein degradation. The atomic details of whole CRL assembly and interactions that dictate subunit specificity remain elusive. Here we present the crystal structure of a pentameric CRL2VHL complex, composed of Cul2, Rbx1, Elongin B, Elongin C, and pVHL. The structure traps a closed state of full-length Cul2 and a new pose of Rbx1 in a trajectory from closed to open conformation. We characterize hotspots and binding thermodynamics at the interface between Cul2 and pVHL-EloBC and identify mutations that contribute toward a selectivity switch for Cul2 versus Cul5 recognition. Our findings provide structural and biophysical insights into the whole Cul2 complex that could aid future drug targeting.
