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

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Basic information

Entry
Database: PDB / ID: 8g46
TitleCryo-EM structure of DDB1deltaB-DDA1-DCAF16-BRD4(BD2)-MMH2
Components
  • Bromodomain-containing protein 4
  • DDB1- and CUL4-associated factor 16
  • DET1- and DDB1-associated protein 1
  • DNA damage-binding protein 1
KeywordsLIGASE / E3 ligase / ubiquitin / degrader / targeted protein degradation
Function / homology
Function and homology information


positive regulation by virus of viral protein levels in host cell / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / 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 / Cul4-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex ...positive regulation by virus of viral protein levels in host cell / spindle assembly involved in female meiosis / epigenetic programming in the zygotic pronuclei / 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 / Cul4-RING E3 ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / negative regulation of reproductive process / RNA polymerase II C-terminal domain binding / negative regulation of developmental process / P-TEFb complex binding / negative regulation of DNA damage checkpoint / histone H4 reader activity / viral release from host cell / cullin family protein binding / host-mediated suppression of viral transcription / positive regulation of G2/M transition of mitotic cell cycle / ectopic germ cell programmed cell death / positive regulation of T-helper 17 cell lineage commitment / positive regulation of viral genome replication / proteasomal protein catabolic process / : / positive regulation of gluconeogenesis / RNA polymerase II CTD heptapeptide repeat kinase activity / condensed nuclear chromosome / transcription coregulator activity / nucleotide-excision repair / positive regulation of transcription elongation by RNA polymerase II / Recognition of DNA damage by PCNA-containing replication complex / regulation of circadian rhythm / DNA Damage Recognition in GG-NER / 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 / p53 binding / cellular response to UV / rhythmic process / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / chromosome / site of double-strand break / Neddylation / regulation of inflammatory response / ubiquitin-dependent protein catabolic process / histone binding / protein-macromolecule adaptor activity / Potential therapeutics for SARS / proteasome-mediated ubiquitin-dependent protein catabolic process / damaged DNA binding / transcription coactivator activity / chromosome, telomeric region / positive regulation of canonical NF-kappaB signal transduction / transcription cis-regulatory region binding / protein ubiquitination / chromatin remodeling / DNA repair / protein serine/threonine kinase activity / apoptotic process / DNA damage response / chromatin binding / regulation of transcription by RNA polymerase II / negative regulation of apoptotic process / chromatin / positive regulation of DNA-templated transcription / protein-containing complex binding / nucleolus / enzyme binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / extracellular space / DNA binding / 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 / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region ...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 / RSE1/DDB1/CPSF1 second beta-propeller / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / : / CPSF A subunit region / RSE1/DDB1/CPSF1 first beta-propeller / Bromodomain protein 4, C-terminal / C-terminal domain of bromodomain protein 4 / Brdt, bromodomain, repeat I / Brdt, bromodomain, repeat II / NET domain superfamily / NET domain profile. / : / NET domain / Bromodomain extra-terminal - transcription regulation / YVTN repeat-like/Quinoprotein amine dehydrogenase / 7 Propeller / Methylamine Dehydrogenase; Chain H / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / bromo domain / Bromodomain / Bromodomain (BrD) profile. / Bromodomain-like superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / Mainly Beta
Similarity search - Domain/homology
Chem-YK3 / 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 speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsMa, M.W. / Hunkeler, M. / Jin, C.Y. / Fischer, E.S.
Funding support United States, 3items
OrganizationGrant numberCountry
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 Foundation19-001-ELA United States
CitationJournal: 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
DepositionFeb 8, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 8, 2023Provider: repository / Type: Initial release
Revision 1.1Oct 23, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_3d_fitting_list.initial_refinement_model_id / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA damage-binding protein 1
B: DDB1- and CUL4-associated factor 16
C: Bromodomain-containing protein 4
E: DET1- and DDB1-associated protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)148,6516
Polymers148,0564
Non-polymers5952
Water1,982110
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8960 Å2
ΔGint-43 kcal/mol
Surface area44590 Å2
MethodPISA

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Components

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Protein , 4 types, 4 molecules ABCE

#1: Protein DNA damage-binding protein 1 / DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / ...DDB p127 subunit / DNA damage-binding protein a / DDBa / Damage-specific DNA-binding protein 1 / HBV X-associated protein 1 / XAP-1 / UV-damaged DNA-binding factor / UV-damaged DNA-binding protein 1 / UV-DDB 1 / XPE-binding factor / XPE-BF / Xeroderma pigmentosum group E-complementing protein / XPCe


Mass: 96425.586 Da / Num. of mol.: 1
Fragment: UNP residues 1-395 + GNGNSG linker + UNP residues 706-1140
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1, XAP1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q16531
#2: Protein DDB1- and CUL4-associated factor 16


Mass: 24547.697 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DCAF16, C4orf30 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9NXF7
#3: Protein Bromodomain-containing protein 4 / Protein HUNK1


Mass: 14899.109 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BRD4, HUNK1 / Production host: Escherichia coli (E. coli) / References: UniProt: O60885
#4: Protein DET1- and DDB1-associated protein 1 / Placenta cross-immune reaction antigen 1 / PCIA-1


Mass: 12183.646 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDA1, C19orf58, PCIA1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q9BW61

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Non-polymers , 3 types, 112 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical 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


Mass: 529.675 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H31N5O4S2 / Feature type: SUBJECT OF INVESTIGATION
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 110 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: 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 / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.148 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Trichoplusia ni (cabbage looper) / Strain: Hi5
Buffer solutionpH: 7.4
Details: 50 mM HEPES pH 7.4, 200 mM NaCl, 2 mM TCEP, 0.011% LMNG
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid1
2200 mMsodium chlorideNaCl1
30.011 %Lauryl Maltose Neopentyl Glycol1
42 mMtris(2-carboxyethyl)phosphine1
SpecimenConc.: 1.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 ...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.
Specimen supportDetails: 20 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R0./1
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 283 K / Details: detergent added directly before grid application

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.3 sec. / Electron dose: 50.27 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 17118
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: REFMAC / Version: 5.8.0352 / Classification: refinement
EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEM4.0.5image acquisition
4cryoSPARC3.3.2CTF correction
7UCSF ChimeraX1.4model fitting
9cryoSPARC3.3.2initial Euler assignment
10cryoSPARC3.3.2final Euler assignment
11cryoSPARC3.3.2classification
12cryoSPARC3.3.23D reconstruction
13ISOLDE1.3model refinement
14REFMAC5.8model refinement
15PHENIX1.19model refinement
CTF correctionDetails: in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 14452363
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1433050 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 74 / Protocol: OTHER / Details: refinement in both real and Fourier space
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDAccession codeDetailsInitial refinement model-ID
26VIX

6vix

6VIXfor BRD4(BD2)1
36Q0R

6q0r

6Q0Rfor DDB1 and DDA12
RefinementResolution: 2.2→146.97 Å / Cor.coef. Fo:Fc: 0.779 / SU B: 4.489 / SU ML: 0.105 / ESU R: 0.165
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.39229 --
obs0.39229 269015 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 73.259 Å2
Refinement stepCycle: 1 / Total: 8603
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.0128677
ELECTRON MICROSCOPYr_bond_other_d00.0168022
ELECTRON MICROSCOPYr_angle_refined_deg1.0711.6511757
ELECTRON MICROSCOPYr_angle_other_deg0.3561.56218725
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.25451063
ELECTRON MICROSCOPYr_dihedral_angle_2_deg2.425548
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.362101531
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.050.21326
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.029710
ELECTRON MICROSCOPYr_gen_planes_other0.0010.021663
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.3897.1214270
ELECTRON MICROSCOPYr_mcbond_other6.3877.124270
ELECTRON MICROSCOPYr_mcangle_it9.39110.6765328
ELECTRON MICROSCOPYr_mcangle_other9.39110.6765329
ELECTRON MICROSCOPYr_scbond_it6.4897.9774407
ELECTRON MICROSCOPYr_scbond_other6.4887.9774408
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other10.07911.5926430
ELECTRON MICROSCOPYr_long_range_B_refined16.57735483
ELECTRON MICROSCOPYr_long_range_B_other16.58135450
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.2→2.257 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.987 19924 -
obs--100 %

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