+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29714 | ||||||||||||
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Title | Cryo-EM structure of DDB1deltaB-DDA1-DCAF16-BRD4(BD2)-MMH2 | ||||||||||||
Map data | main map | ||||||||||||
Sample |
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Function / homology | Function and homology information 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 / regulation of mitotic cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / WD40-repeat domain binding / ubiquitin ligase complex scaffold activity ...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 / regulation of mitotic cell cycle phase transition / Cul4A-RING E3 ubiquitin ligase complex / WD40-repeat domain binding / ubiquitin ligase complex scaffold activity / Cul4B-RING E3 ubiquitin ligase complex / negative regulation of reproductive process / negative regulation of developmental process / cullin family protein binding / viral release from host cell / ectopic germ cell programmed cell death / proteasomal protein catabolic process / positive regulation of viral genome replication / positive regulation of gluconeogenesis / regulation of circadian rhythm / nucleotide-excision repair / Recognition of DNA damage by PCNA-containing replication complex / DNA Damage Recognition in GG-NER / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / Formation of TC-NER Pre-Incision Complex / Dual Incision in GG-NER / 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 / rhythmic process / cellular response to UV / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / Neddylation / protein-macromolecule adaptor activity / site of double-strand break / chromosome, telomeric region / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / toxin activity / damaged DNA binding / protein ubiquitination / DNA repair / DNA damage response / negative regulation of apoptotic process / protein-containing complex binding / nucleolus / apoptotic process / protein-containing complex / DNA binding / extracellular space / extracellular exosome / nucleoplasm / nucleus / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Homo sapiens (human) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.2 Å | ||||||||||||
Authors | Ma MW / Hunkeler M / Jin CY / Fischer ES | ||||||||||||
Funding support | United States, 3 items
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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. | ||||||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29714.map.gz | 97.1 MB | EMDB map data format | |
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Header (meta data) | emd-29714-v30.xml emd-29714.xml | 29 KB 29 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_29714_fsc.xml | 9.8 KB | Display | FSC data file |
Images | emd_29714.png | 78.7 KB | ||
Masks | emd_29714_msk_1.map | 103 MB | Mask map | |
Others | emd_29714_additional_1.map.gz emd_29714_half_map_1.map.gz emd_29714_half_map_2.map.gz | 90 MB 95.7 MB 95.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29714 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29714 | HTTPS FTP |
-Validation report
Summary document | emd_29714_validation.pdf.gz | 853.9 KB | Display | EMDB validaton report |
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Full document | emd_29714_full_validation.pdf.gz | 853.4 KB | Display | |
Data in XML | emd_29714_validation.xml.gz | 18.4 KB | Display | |
Data in CIF | emd_29714_validation.cif.gz | 23.7 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29714 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29714 | HTTPS FTP |
-Related structure data
Related structure data | 8g46MC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_29714.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | main map | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.88533 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_29714_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: map post-processed with deepEMhancer
File | emd_29714_additional_1.map | ||||||||||||
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Annotation | map post-processed with deepEMhancer | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map B
File | emd_29714_half_map_1.map | ||||||||||||
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Annotation | half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map A
File | emd_29714_half_map_2.map | ||||||||||||
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Annotation | half map A | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-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 |
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Components |
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-Supramolecule #1: Ternary complex of cullin ring E3 ubiquitin ligase substrate rece...
Supramolecule | Name: Ternary complex of cullin ring E3 ubiquitin ligase substrate receptor arm (DDB1deltaB-DDA1-DCAF16) in compound-induced complex with bromodomain 2 of BRD4 type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 148 KDa |
-Macromolecule #1: DNA damage-binding protein 1
Macromolecule | Name: DNA damage-binding protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 96.425586 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: MGSSHHHHHH SAAHIVMVDA YKPTKGGRMS YNYVVTAQKP TAVNGCVTGH FTSAEDLNLL IAKNTRLEIY VVTAEGLRPV KEVGMYGKI AVMELFRPKG ESKDLLFILT AKYNACILEY KQSGESIDII TRAHGNVQDR IGRPSETGII GIIDPECRMI G LRLYDGLF ...String: MGSSHHHHHH SAAHIVMVDA YKPTKGGRMS YNYVVTAQKP TAVNGCVTGH FTSAEDLNLL IAKNTRLEIY VVTAEGLRPV KEVGMYGKI AVMELFRPKG ESKDLLFILT AKYNACILEY KQSGESIDII TRAHGNVQDR IGRPSETGII GIIDPECRMI G LRLYDGLF KVIPLDRDNK ELKAFNIRLE ELHVIDVKFL YGCQAPTICF VYQDPQGRHV KTYEVSLREK EFNKGPWKQE NV EAEASMV IAVPEPFGGA IIIGQESITY HNGDKYLAIA PPIIKQSTIV CHNRVDPNGS RYLLGDMEGR LFMLLLEKEE QMD GTVTLK DLRVELLGET SIAECLTYLD NGVVFVGSRL GDSQLVKLNV DSNEQGSYVV AMETFTNLGP IVDMCVVDLE RQGQ GQLVT CSGAFKEGSL RIIRNGIGGN GNSGEIQKLH IRTVPLYESP RKICYQEVSQ CFGVLSSRIE VQDTSGGTTA LRPSA STQA LSSSVSSSKL FSSSTAPHET SFGEEVEVHN LLIIDQHTFE VLHAHQFLQN EYALSLVSCK LGKDPNTYFI VGTAMV YPE EAEPKQGRIV VFQYSDGKLQ TVAEKEVKGA VYSMVEFNGK LLASINSTVR LYEWTTEKEL RTECNHYNNI MALYLKT KG DFILVGDLMR SVLLLAYKPM EGNFEEIARD FNPNWMSAVE ILDDDNFLGA ENAFNLFVCQ KDSAATTDEE RQHLQEVG L FHLGEFVNVF CHGSLVMQNL GETSTPTQGS VLFGTVNGMI GLVTSLSESW YNLLLDMQNR LNKVIKSVGK IEHSFWRSF HTERKTEPAT GFIDGDLIES FLDISRPKMQ EVVANLQYDD GSGMKREATA DDLIKVVEEL TRIH |
-Macromolecule #2: DDB1- and CUL4-associated factor 16
Macromolecule | Name: DDB1- and CUL4-associated factor 16 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 24.547697 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: GGGRMGPRNP SPDHLSESES EEEENISYLN ESSGEEWDSS EEEDSMVPNL SPLESLAWQV KCLLKYSTTW KPLNPNSWLY HAKLLDPST PVHILREIGL RLSHCSHCVP KLEPIPEWPP LASCGVPPFQ KPLTSPSRLS RDHATLNGAL QFATKQLSRT L SRATPIPE ...String: GGGRMGPRNP SPDHLSESES EEEENISYLN ESSGEEWDSS EEEDSMVPNL SPLESLAWQV KCLLKYSTTW KPLNPNSWLY HAKLLDPST PVHILREIGL RLSHCSHCVP KLEPIPEWPP LASCGVPPFQ KPLTSPSRLS RDHATLNGAL QFATKQLSRT L SRATPIPE YLKQIPNSCV SGCCCGWLTK TVKETTRTEP INTTYSYTDF QKAVNKLLTA SL |
-Macromolecule #3: Bromodomain-containing protein 4
Macromolecule | Name: Bromodomain-containing protein 4 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 14.899109 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GKDVPDSQQH PAPEKSSKVS EQLKCCSGIL KEMFAKKHAA YAWPFYKPVD VEALGLHDYC DIIKHPMDMS TIKSKLEARE YRDAQEFGA DVRLMFSNCY KYNPPDHEVV AMARKLQDVF EMRFAKMPDE |
-Macromolecule #4: DET1- and DDB1-associated protein 1
Macromolecule | Name: DET1- and DDB1-associated protein 1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 12.183646 KDa |
Recombinant expression | Organism: Trichoplusia ni (cabbage looper) |
Sequence | String: GGGRMADFLK GLPVYNKSNF SRFHADSVCK ASNRRPSVYL PTREYPSEQI IVTEKTNILL RYLHQQWDKK NAAKKRDQEQ VELEGESSA PPRKVARTDS PDMHEDT |
-Macromolecule #5: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Macromolecule #6: tert-butyl [(6S,10P)-4-{4-[(ethanesulfonyl)amino]phenyl}-2,3,9-tr...
Macromolecule | Name: tert-butyl [(6S,10P)-4-{4-[(ethanesulfonyl)amino]phenyl}-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetate type: ligand / ID: 6 / Number of copies: 1 / Formula: YK3 |
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Molecular weight | Theoretical: 529.675 Da |
Chemical component information | ChemComp-YK3: |
-Macromolecule #7: water
Macromolecule | Name: water / type: ligand / ID: 7 / Number of copies: 110 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.25 mg/mL | |||||||||||||||
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Buffer | pH: 7.4 Component:
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. |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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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 |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |