EMDB-29714: Cryo-EM structure of DDB1deltaB-DDA1-DCAF16-BRD4(BD2)-MMH2

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 4BRD4
  • Protein or peptide: DET1- and DDB1-associated protein 1
• Ligand: ZINC ION
• Ligand: 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
• Ligand: water

Function / homology

Function:

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 / WD40-repeat domain binding / 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 / positive regulation of T-helper 17 cell lineage commitment / positive regulation of viral genome replication / 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 / proteasomal protein catabolic process / 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 / protein polyubiquitination / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / positive regulation of protein catabolic process / cellular response to UV / rhythmic process / protein-macromolecule adaptor activity / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / p53 binding / site of double-strand break / Neddylation / chromosome / 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 / protein ubiquitination / transcription cis-regulatory region binding / chromatin remodeling / DNA repair / apoptotic process / DNA damage response / chromatin binding / protein-containing complex binding / 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

Domain/homology:

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

Component:

Bromodomain-containing protein 4 / DNA damage-binding protein 1 / DET1- and DDB1-associated protein 1 / DDB1- and CUL4-associated factor 16

• 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

Organization	Grant number	Country
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 / 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 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

Deposition	Feb 8, 2023	-
Header (metadata) release	Mar 8, 2023	-
Map release	Mar 8, 2023	-
Update	Mar 8, 2023	-
Current status	Mar 8, 2023	Processing site: RCSB / Status: Released

Map

• File: Download / File: emd_29714.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: main map
• Voxel size: X=Y=Z: 0.88533 Å

Density

Contour Level	By AUTHOR: 0.463
Minimum - Maximum	-2.0747068 - 3.0200558
Average (Standard dev.)	-0.00020515308 (±0.070798434)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order X Y Z Origin 0 0 0 Dimensions 300 300 300 Spacing 300 300 300
Cell	A=B=C: 265.599 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_29714_msk_1.map

Additional map: map post-processed with deepEMhancer

• File: emd_29714_additional_1.map
• Annotation: map post-processed with deepEMhancer

Half map: half map B

• File: emd_29714_half_map_1.map
• Annotation: half map B

Half map: half map A

• File: emd_29714_half_map_2.map
• Annotation: half map A

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

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 4BRD4
  • Protein or peptide: DET1- and DDB1-associated protein 1
• Ligand: ZINC ION
• Ligand: 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
• Ligand: water

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
• 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
• 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 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
• 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 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
• 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
• 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
• 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
• Molecular weight: Theoretical: 529.675 Da

Chemical component information

ChemComp-YK3:
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

Macromolecule #7: water

• Macromolecule: Name: water / type: ligand / ID: 7 / Number of copies: 110 / Formula: HOH
• Molecular weight: Theoretical: 18.015 Da

Chemical component information

ChemComp-HOH:
WATER / Water

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 1.25 mg/mL

Buffer

pH: 7.4
Component:

Concentration	Name	Formula
50.0 mM	4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
200.0 mM	sodium chloride	NaClSodium chloride
0.011 %	Lauryl Maltose Neopentyl Glycol
2.0 mM	tris(2-carboxyethyl)phosphine

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
• 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 FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
• Sample stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• 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/Ų
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 14452363
• Startup model: Type of model: NONE / Details: built de novo
• Initial angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
• Final 3D classification: Number classes: 8 / Avg.num./class: 600000 / Software - Name: cryoSPARC (ver. 3.3.2) / Details: 3D variability followed by clustering in cryoSPARC
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
• Final reconstruction: Number classes used: 3 / Applied symmetry - Point group: C₁ (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 1433050

Atomic model buiding 1

Initial model

PDB ID	Details
6vix	for BRD4(BD2)
6q0r	for DDB1 and DDA1

• Details: refinement in both real and Fourier space
• Refinement: Protocol: OTHER / Overall B value: 74

Output model

PDB-8g46:
Cryo-EM structure of DDB1deltaB-DDA1-DCAF16-BRD4(BD2)-MMH2