RESUMO
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.
Assuntos
DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Elonguina/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , RNA Polimerase II/genética , Ubiquitina-Proteína Ligases/genética , Animais , Síndrome de Cockayne/enzimologia , Síndrome de Cockayne/genética , DNA Helicases/química , DNA Helicases/ultraestrutura , Reparo do DNA/genética , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/ultraestrutura , Elonguina/química , Elonguina/ultraestrutura , Humanos , Camundongos , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas de Ligação a Poli-ADP-Ribose/química , Proteínas de Ligação a Poli-ADP-Ribose/ultraestrutura , RNA Polimerase II/química , Receptores de Glucocorticoides/química , Receptores de Glucocorticoides/genética , Ubiquitina/química , Ubiquitina/genética , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/ultraestrutura , Ubiquitinação/genéticaRESUMO
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.
Assuntos
Proteínas Culina/química , Citidina Desaminase/química , Simulação de Dinâmica Molecular , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Desaminases APOBEC , Subunidade beta de Fator de Ligação ao Core/química , Cristalização , Elonguina/química , Humanos , Cinética , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Relação Estrutura-Atividade , UbiquitinaçãoRESUMO
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.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Culina/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Elonguina/química , Elonguina/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de ProteínaRESUMO
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.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Complexos Multiproteicos/química , Ubiquitina-Proteína Ligases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Elonguina/química , Elonguina/metabolismo , Fluorometria/métodos , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Complexos Multiproteicos/metabolismo , Policitemia/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitina-Proteína Ligases/química , Proteína Supressora de Tumor Von Hippel-Lindau/químicaRESUMO
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.
