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- PDB-9c5v: Cryo EM structure of a DCAF2:degrader:BRD4 ternary complex -

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Basic information

Entry
Database: PDB / ID: 9c5v
TitleCryo EM structure of a DCAF2:degrader:BRD4 ternary complex
Components
  • Bromodomain-containing protein 4
  • DET1- and DDB1-associated protein 1
  • DNA damage-binding protein 1
  • Denticleless protein homolog
KeywordsLIGASE / DTL / DDB1 / DDA1 / BRD4 / degrader / covalent
Function / homology
Function and homology information


regulation of cell cycle phase transition / regulation of natural killer cell activation / 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 / regulation of DNA-templated DNA replication initiation / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding ...regulation of cell cycle phase transition / regulation of natural killer cell activation / 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 / regulation of DNA-templated DNA replication initiation / biological process involved in interaction with symbiont / regulation of mitotic cell cycle phase transition / WD40-repeat domain binding / mitotic G2 DNA damage checkpoint signaling / histone H4K8ac reader activity / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / negative regulation of reproductive process / negative regulation of developmental process / Cul4B-RING E3 ubiquitin ligase complex / ectopic germ cell programmed cell death / RNA polymerase II C-terminal domain binding / histone H3K27ac reader activity / negative regulation of DNA damage checkpoint / ubiquitin ligase complex scaffold activity / P-TEFb complex binding / histone H3K9ac reader activity / histone H4 reader activity / histone H4K5ac reader activity / histone H4K12ac reader activity / host-mediated suppression of viral transcription / viral release from host cell / histone H4K16ac reader activity / positive regulation of G2/M transition of mitotic cell cycle / protein monoubiquitination / response to UV / cullin family protein binding / positive regulation of T-helper 17 cell lineage commitment / positive regulation of viral genome replication / translesion synthesis / positive regulation of gluconeogenesis / RNA polymerase II CTD heptapeptide repeat kinase activity / condensed nuclear chromosome / sperm principal piece / sperm end piece / transcription coregulator activity / nucleotide-excision repair / proteasomal protein catabolic process / positive regulation of transcription elongation by RNA polymerase II / Recognition of DNA damage by PCNA-containing replication complex / regulation of circadian rhythm / Wnt signaling pathway / 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 / protein polyubiquitination / Formation of Incision Complex in GG-NER / sperm midpiece / positive regulation of protein catabolic process / cellular response to UV / p53 binding / Regulation of PD-L1(CD274) transcription / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / rhythmic process / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / regulation of inflammatory response / chromosome / site of double-strand break / nuclear membrane / Neddylation / histone binding / Potential therapeutics for SARS / damaged DNA binding / ubiquitin-dependent protein catabolic process / proteasome-mediated ubiquitin-dependent protein catabolic process / regulation of cell cycle / positive regulation of canonical NF-kappaB signal transduction / chromosome, telomeric region / protein-macromolecule adaptor activity / transcription coactivator activity / transcription cis-regulatory region binding / protein ubiquitination / chromatin remodeling / DNA repair / protein serine/threonine kinase activity / centrosome / chromatin binding / apoptotic process / regulation of transcription by RNA polymerase II / DNA damage response / nucleolus / negative regulation of apoptotic process / positive regulation of DNA-templated transcription / chromatin / protein-containing complex binding / enzyme binding / positive regulation of transcription by RNA polymerase II / protein-containing complex / DNA-templated transcription / DNA binding / :
Similarity search - Function
: / 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 ...: / 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 / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / bromo domain / Bromodomain / Bromodomain (BrD) profile. / Bromodomain-like superfamily / WD domain, G-beta repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40 repeats / WD40 repeat / 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 / Denticleless protein homolog
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.36 Å
AuthorsMcMahon, E.J. / Wang, W.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Structure / Year: 2025
Title: Structural basis for DCAF2 as a novel E3 ligase for PROTAC-mediated targeted protein degradation.
Authors: Evan J McMahon / Alexander G Cioffi / Patrick R Visperas / Yueqing Lin / Michael Shaghafi / Courtney M Daczkowski / Johannes C Hermann / Robert A Everley / Richard M Neve / Daniel A Erlanson ...Authors: Evan J McMahon / Alexander G Cioffi / Patrick R Visperas / Yueqing Lin / Michael Shaghafi / Courtney M Daczkowski / Johannes C Hermann / Robert A Everley / Richard M Neve / Daniel A Erlanson / Kevin R Webster / Vikram Narayan / Weiru Wang /
Abstract: Targeted protein degradation (TPD) leverages the ubiquitin-proteasome system to eliminate disease-causing proteins via E3 ligases. To date, the field is limited to utilizing a few of the over 600 ...Targeted protein degradation (TPD) leverages the ubiquitin-proteasome system to eliminate disease-causing proteins via E3 ligases. To date, the field is limited to utilizing a few of the over 600 human E3 ligases. To expand this repertoire, we conducted structural and functional validation of DDB1 (Damage-specific DNA binding protein 1) and Cullin-associated factor (DCAF)2 (DTL/CDT2), a Cullin4-RING ligase substrate adaptor implicated in DNA damage response and cancer, as a novel E3 for TPD. Cryoelectron microscopy (cryo-EM) structures of the DCAF2:DDB1:DDA1 complex (3.3 Å), a ligand bound complex (3.1 Å), and a ternary complex with a covalent proteolysis-targeting chimera (PROTAC) and BRD4 (3.4 Å) reveal PROTAC-mediated substrate recruitment. Using covalent bifunctional tool compounds engaging residue C141 in the WD40 domain, we demonstrate robust ubiquitination in biochemical assays and cellular TPD using the COFFEE (covalent functionalization followed by E3 electroporation) method. These findings position DCAF2 as a promising E3 adaptor for PROTAC strategies and identify C141 as a relevant site for future PROTAC discovery.
History
DepositionJun 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 1, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Denticleless protein homolog
B: DNA damage-binding protein 1
D: Bromodomain-containing protein 4
C: DET1- and DDB1-associated protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)207,2895
Polymers206,4094
Non-polymers8801
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Denticleless protein homolog / DDB1- and CUL4-associated factor 2 / Lethal(2) denticleless protein homolog / Retinoic acid- ...DDB1- and CUL4-associated factor 2 / Lethal(2) denticleless protein homolog / Retinoic acid-regulated nuclear matrix-associated protein


Mass: 51887.121 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DTL, CDT2, CDW1, DCAF2, L2DTL, RAMP / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NZJ0
#2: 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: 127097.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DDB1, XAP1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q16531
#3: Protein Bromodomain-containing protein 4 / Protein HUNK1


Mass: 15568.838 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: 11855.297 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BW61
#5: Chemical ChemComp-A1AUO / N-({4-[(4-{[1'-(chloroacetyl)-3'-oxo-3',4'-dihydro-1'H-spiro[cyclopentane-1,2'-quinoxalin]-6'-yl]oxy}piperidin-1-yl)methyl]phenyl}methyl)-2-[(6S,10P)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl]acetamide


Mass: 879.896 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C46H48Cl2N8O4S / Feature type: SUBJECT OF INVESTIGATION
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: DCAF2:degrader:BRD4 ternary complex / Type: COMPLEX / Entity ID: #1-#2, #4, #3 / Source: RECOMBINANT
Molecular weightValue: 0.206 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.4 sec. / Electron dose: 50.21 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1
EM imaging opticsPhase plate: VOLTA PHASE PLATE

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9PHENIX1.20.1_4487:model refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1277208
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70550 / Symmetry type: POINT
RefinementHighest resolution: 3.36 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413241
ELECTRON MICROSCOPYf_angle_d0.83217946
ELECTRON MICROSCOPYf_dihedral_angle_d11.3591815
ELECTRON MICROSCOPYf_chiral_restr0.0472009
ELECTRON MICROSCOPYf_plane_restr0.0052307

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