- EMDB-29714: Cryo-EM structure of DDB1deltaB-DDA1-DCAF16-BRD4(BD2)-MMH2 -
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Basic information
Entry
Database: EMDB / ID: EMD-29714
Title
Cryo-EM structure of DDB1deltaB-DDA1-DCAF16-BRD4(BD2)-MMH2
Map data
main map
Sample
Complex: Ternary complex of cullin ring E3 ubiquitin ligase substrate receptor arm (DDB1deltaB-DDA1-DCAF16) in compound-induced complex with bromodomain 2 of BRD4
Protein or peptide: DNA damage-binding protein 1
Protein or peptide: DDB1- and CUL4-associated factor 16
Protein or peptide: Bromodomain-containing protein 4
Protein or peptide: DET1- and DDB1-associated protein 1
positive regulation by virus of viral protein levels in host cell / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / biological process involved in interaction with symbiont / WD40-repeat domain binding / regulation of mitotic cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex ...positive regulation by virus of viral protein levels in host cell / epigenetic programming in the zygotic pronuclei / spindle assembly involved in female meiosis / Cul4-RING E3 ubiquitin ligase complex / UV-damage excision repair / biological process involved in interaction with symbiont / WD40-repeat domain binding / regulation of mitotic cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of reproductive process / negative regulation of developmental process / cullin family protein binding / RNA polymerase II C-terminal domain binding / negative regulation of DNA damage checkpoint / P-TEFb complex binding / viral release from host cell / negative regulation by host of viral transcription / ectopic germ cell programmed cell death / proteasomal protein catabolic process / positive regulation of viral genome replication / positive regulation of T-helper 17 cell lineage commitment / positive regulation of gluconeogenesis / positive regulation of G2/M transition of mitotic cell cycle / histone reader activity / RNA polymerase II CTD heptapeptide repeat kinase activity / condensed nuclear chromosome / nucleotide-excision repair / Recognition of DNA damage by PCNA-containing replication complex / positive regulation of transcription elongation by RNA polymerase II / transcription coregulator activity / DNA Damage Recognition in GG-NER / lysine-acetylated histone binding / regulation of circadian rhythm / Dual Incision in GG-NER / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Wnt signaling pathway / Formation of Incision Complex in GG-NER / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / protein polyubiquitination / positive regulation of protein catabolic process / cellular response to UV / rhythmic process / p53 binding / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / protein-macromolecule adaptor activity / chromosome / Neddylation / site of double-strand break / regulation of inflammatory response / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / positive regulation of canonical NF-kappaB signal transduction / Potential therapeutics for SARS / chromosome, telomeric region / damaged DNA binding / transcription coactivator activity / transcription cis-regulatory region binding / protein ubiquitination / chromatin remodeling / DNA repair / apoptotic process / DNA damage response / chromatin binding / protein-containing complex binding / chromatin / nucleolus / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / positive regulation of DNA-templated transcription / enzyme binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA binding / extracellular space / extracellular exosome / nucleoplasm / nucleus / cytoplasm Similarity search - Function
DDB1- and CUL4-associated factor 16 / DDB1- and CUL4-associated factor 16 / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / Det1 complexing ubiquitin ligase / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / Bromodomain protein 4, C-terminal ...DDB1- and CUL4-associated factor 16 / DDB1- and CUL4-associated factor 16 / DET1- and DDB1-associated protein 1, N-terminal / DET1- and DDB1-associated protein 1 / Det1 complexing ubiquitin ligase / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / Bromodomain protein 4, C-terminal / C-terminal domain of bromodomain protein 4 / NET domain superfamily / NET domain profile. / Brdt, bromodomain, repeat II / Brdt, bromodomain, repeat I / NET domain / Bromodomain extra-terminal - transcription regulation / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / Bromodomain profile. / bromo domain / Bromodomain / Bromodomain-like superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily Similarity search - Domain/homology
Bromodomain-containing protein 4 / DNA damage-binding protein 1 / DET1- and DDB1-associated protein 1 / DDB1- and CUL4-associated factor 16 Similarity search - Component
Biological species
Homo sapiens (human)
Method
single particle reconstruction / cryo EM / Resolution: 2.2 Å
National Institutes of Health/National Cancer Institute (NIH/NCI)
CA262188
United States
National Institutes of Health/National Cancer Institute (NIH/NCI)
CA066996
United States
The Mark Foundation
19-001-ELA
United States
Citation
Journal: bioRxiv / Year: 2023 Title: Template-assisted covalent modification of DCAF16 underlies activity of BRD4 molecular glue degraders. Authors: Yen-Der Li / Michelle W Ma / Muhammad Murtaza Hassan / Moritz Hunkeler / Mingxing Teng / Kedar Puvar / Ryan Lumpkin / Brittany Sandoval / Cyrus Y Jin / Scott B Ficarro / Michelle Y Wang / ...Authors: Yen-Der Li / Michelle W Ma / Muhammad Murtaza Hassan / Moritz Hunkeler / Mingxing Teng / Kedar Puvar / Ryan Lumpkin / Brittany Sandoval / Cyrus Y Jin / Scott B Ficarro / Michelle Y Wang / Shawn Xu / Brian J Groendyke / Logan H Sigua / Isidoro Tavares / Charles Zou / Jonathan M Tsai / Paul M C Park / Hojong Yoon / Felix C Majewski / Jarrod A Marto / Jun Qi / Radosław P Nowak / Katherine A Donovan / Mikołaj Słabicki / Nathanael S Gray / Eric S Fischer / Benjamin L Ebert / Abstract: Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics. Molecular glues are of ...Small molecules that induce protein-protein interactions to exert proximity-driven pharmacology such as targeted protein degradation are a powerful class of therapeutics. Molecular glues are of particular interest given their favorable size and chemical properties and represent the only clinically approved degrader drugs. The discovery and development of molecular glues for novel targets, however, remains challenging. Covalent strategies could in principle facilitate molecular glue discovery by stabilizing the neo-protein interfaces. Here, we present structural and mechanistic studies that define a -labeling covalent molecular glue mechanism, which we term "template-assisted covalent modification". We found that a novel series of BRD4 molecular glue degraders act by recruiting the CUL4 ligase to the second bromodomain of BRD4 (BRD4). BRD4, in complex with DCAF16, serves as a structural template to facilitate covalent modification of DCAF16, which stabilizes the BRD4-degrader-DCAF16 ternary complex formation and facilitates BRD4 degradation. A 2.2 Å cryo-electron microscopy structure of the ternary complex demonstrates that DCAF16 and BRD4 have pre-existing structural complementarity which optimally orients the reactive moiety of the degrader for DCAF16 covalent modification. Systematic mutagenesis of both DCAF16 and BRD4 revealed that the loop conformation around BRD4, rather than specific side chains, is critical for stable interaction with DCAF16 and BD2 selectivity. Together our work establishes "template-assisted covalent modification" as a mechanism for covalent molecular glues, which opens a new path to proximity driven pharmacology.
Entire : Ternary complex of cullin ring E3 ubiquitin ligase substrate rece...
Entire
Name: Ternary complex of cullin ring E3 ubiquitin ligase substrate receptor arm (DDB1deltaB-DDA1-DCAF16) in compound-induced complex with bromodomain 2 of BRD4
Components
Complex: Ternary complex of cullin ring E3 ubiquitin ligase substrate receptor arm (DDB1deltaB-DDA1-DCAF16) in compound-induced complex with bromodomain 2 of BRD4
Protein or peptide: DNA damage-binding protein 1
Protein or peptide: DDB1- and CUL4-associated factor 16
Protein or peptide: Bromodomain-containing protein 4
Protein or peptide: DET1- and DDB1-associated protein 1
Details: 50 mM HEPES pH 7.4, 200 mM NaCl, 2 mM TCEP, 0.011% LMNG
Grid
Model: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 40.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 3.9e-05 kPa / Details: 20 mA
Vitrification
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 283 K / Instrument: LEICA EM GP / Details: detergent added directly before grid application.
Details
The ternary complex was incubated at RT for 30 min before polishing on through size exclusion chromatography. The purified DCAF16 complex was incubated with an extra 1.2x molar excess of purified BRD4BD2 for 30 minutes at 4 degree C before grid preparation.
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Electron microscopy
Microscope
FEI TITAN KRIOS
Specialist optics
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recording
Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 17118 / Average exposure time: 2.3 sec. / Average electron dose: 50.27 e/Å2
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
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