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- EMDB-51338: Cryo-EM structure of KBTBD4 WT-HDAC2 2:2 complex mediated by mole... -

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Entry
Database: EMDB / ID: EMD-51338
TitleCryo-EM structure of KBTBD4 WT-HDAC2 2:2 complex mediated by molecular glue UM171
Map dataFSC-calculated nominal resolution is 2.9 angstrom. Practical resolution on manual inspection is closer to 4 angstrom in some local areas. The DeepEMhancer map was generated and used for model building
Sample
  • Complex: KBTBD4 WT-HDAC2 2:2 complex mediated by molecular glue UM171
    • Protein or peptide: Isoform 1 of Kelch repeat and BTB domain-containing protein 4
    • Protein or peptide: Histone deacetylase 2
  • Ligand: (1r,4r)-N~1~-[(7P)-2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine
  • Ligand: ZINC ION
KeywordsE3 ligase / deacetylase / ubiquitylation / transcription / molecular glue / complex / LIGASE
Function / homology
Function and homology information


positive regulation of male mating behavior / protein de-2-hydroxyisobutyrylase activity / negative regulation of peptidyl-lysine acetylation / protein lysine delactylase activity / p75NTR negatively regulates cell cycle via SC1 / epidermal cell differentiation / negative regulation of dendritic spine development / histone decrotonylase activity / fungiform papilla formation / negative regulation of MHC class II biosynthetic process ...positive regulation of male mating behavior / protein de-2-hydroxyisobutyrylase activity / negative regulation of peptidyl-lysine acetylation / protein lysine delactylase activity / p75NTR negatively regulates cell cycle via SC1 / epidermal cell differentiation / negative regulation of dendritic spine development / histone decrotonylase activity / fungiform papilla formation / negative regulation of MHC class II biosynthetic process / : / behavioral response to ethanol / positive regulation of interleukin-1 production / NuRD complex / negative regulation of transcription by competitive promoter binding / regulation of cell fate specification / EGR2 and SOX10-mediated initiation of Schwann cell myelination / negative regulation of stem cell population maintenance / ESC/E(Z) complex / histone H4K16 deacetylase activity, hydrolytic mechanism / histone H4K5 deacetylase activity, hydrolytic mechanism / histone H4K8 deacetylase activity, hydrolytic mechanism / histone H3K4 deacetylase activity, hydrolytic mechanism / histone H3K14 deacetylase activity, hydrolytic mechanism / histone H4K12 deacetylase activity, hydrolytic mechanism / cellular response to dopamine / regulation of stem cell differentiation / histone deacetylase / STAT3 nuclear events downstream of ALK signaling / cardiac muscle hypertrophy / histone H3K9 deacetylase activity, hydrolytic mechanism / tubulin deacetylase activity / protein lysine deacetylase activity / response to caffeine / Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In linear amides / embryonic digit morphogenesis / histone deacetylase activity / Notch-HLH transcription pathway / positive regulation of stem cell population maintenance / eyelid development in camera-type eye / Sin3-type complex / dendrite development / odontogenesis of dentin-containing tooth / response to amyloid-beta / positive regulation of proteolysis / RNA Polymerase I Transcription Initiation / Regulation of MECP2 expression and activity / histone deacetylase complex / positive regulation of oligodendrocyte differentiation / hair follicle placode formation / positive regulation of collagen biosynthetic process / NF-kappaB binding / response to hyperoxia / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / Transcriptional regulation of brown and beige adipocyte differentiation by EBF2 / positive regulation of epithelial to mesenchymal transition / MECP2 regulates neuronal receptors and channels / cellular response to transforming growth factor beta stimulus / cellular response to retinoic acid / Regulation of TP53 Activity through Acetylation / heat shock protein binding / response to amphetamine / SUMOylation of chromatin organization proteins / negative regulation of cell migration / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / Regulation of PTEN gene transcription / Regulation of endogenous retroelements by KRAB-ZFP proteins / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / promoter-specific chromatin binding / HDACs deacetylate histones / response to nicotine / negative regulation of transforming growth factor beta receptor signaling pathway / circadian regulation of gene expression / negative regulation of DNA-binding transcription factor activity / response to cocaine / protein modification process / NoRC negatively regulates rRNA expression / NOTCH1 Intracellular Domain Regulates Transcription / cellular response to hydrogen peroxide / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / histone deacetylase binding / heterochromatin formation / positive regulation of tumor necrosis factor production / negative regulation of neuron projection development / Factors involved in megakaryocyte development and platelet production / cellular response to heat / RNA polymerase II-specific DNA-binding transcription factor binding / response to lipopolysaccharide / histone binding / Potential therapeutics for SARS / chromosome, telomeric region / chromatin remodeling / response to xenobiotic stimulus / negative regulation of DNA-templated transcription / positive regulation of cell population proliferation / chromatin binding / negative regulation of apoptotic process / chromatin / positive regulation of DNA-templated transcription
Similarity search - Function
Kelch repeat and BTB domain-containing protein 4 / KBTBD4, BTB/POZ domain / KBTBD4, BACK domain / Kelch repeat type 2 / Kelch motif / Histone deacetylase / BTB-kelch protein / BTB/Kelch-associated / BTB And C-terminal Kelch / BTB And C-terminal Kelch ...Kelch repeat and BTB domain-containing protein 4 / KBTBD4, BTB/POZ domain / KBTBD4, BACK domain / Kelch repeat type 2 / Kelch motif / Histone deacetylase / BTB-kelch protein / BTB/Kelch-associated / BTB And C-terminal Kelch / BTB And C-terminal Kelch / Kelch-type beta propeller / : / Histone deacetylase family / Histone deacetylase domain / Histone deacetylase domain superfamily / Histone deacetylase domain / Ureohydrolase domain superfamily / BTB/POZ domain / BTB domain profile. / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily
Similarity search - Domain/homology
Histone deacetylase 2 / Kelch repeat and BTB domain-containing protein 4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsChen Z / Chi G / Pike ACW / Montes B / Bullock AN
Funding support United Kingdom, Switzerland, 2 items
OrganizationGrant numberCountry
Cancer Research UKDRCNPG-May21/100002 United Kingdom
Innovative Medicines Initiative875510 Switzerland
CitationJournal: Nat Commun / Year: 2025
Title: Structural mimicry of UM171 and neomorphic cancer mutants co-opts E3 ligase KBTBD4 for HDAC1/2 recruitment.
Authors: Zhuoyao Chen / Gamma Chi / Timea Balo / Xiangrong Chen / Beatriz Ralsi Montes / Steven C Clifford / Vincenzo D'Angiolella / Timea Szabo / Arpad Kiss / Tibor Novak / András Herner / András ...Authors: Zhuoyao Chen / Gamma Chi / Timea Balo / Xiangrong Chen / Beatriz Ralsi Montes / Steven C Clifford / Vincenzo D'Angiolella / Timea Szabo / Arpad Kiss / Tibor Novak / András Herner / András Kotschy / Alex N Bullock /
Abstract: Neomorphic mutations and drugs can elicit unanticipated effects that require mechanistic understanding to inform clinical practice. Recurrent indel mutations in the Kelch domain of the KBTBD4 E3 ...Neomorphic mutations and drugs can elicit unanticipated effects that require mechanistic understanding to inform clinical practice. Recurrent indel mutations in the Kelch domain of the KBTBD4 E3 ligase rewire epigenetic programs for stemness in medulloblastoma by recruiting LSD1-CoREST-HDAC1/2 complexes as neo-substrates for ubiquitination and degradation. UM171, an investigational drug for haematopoietic stem cell transplantation, was found to degrade LSD1-CoREST-HDAC1/2 complexes in a wild-type KBTBD4-dependent manner, suggesting a potential common mode of action. Here, we identify that these neomorphic interactions are mediated by the HDAC deacetylase domain. Cryo-EM studies of both wild-type and mutant KBTBD4 capture 2:1 and 2:2 KBTBD4-HDAC2 complexes, as well as a 2:1:1 KBTBD4-HDAC2-CoREST1 complex, at resolutions spanning 2.7 to 3.3 Å. The mutant and drug-induced complexes adopt similar structural assemblies requiring both Kelch domains in the KBTBD4 dimer for each HDAC2 interaction. UM171 is identified as a bona fide molecular glue binding across the ternary interface. Most strikingly, the indel mutation reshapes the same surface of KBTBD4 providing an example of a natural mimic of a molecular glue. Together, the structures provide mechanistic understanding of neomorphic KBTBD4, while structure-activity relationship (SAR) analysis of UM171 reveals analog S234984 as a more potent molecular glue for future studies.
History
DepositionAug 13, 2024-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateApr 9, 2025-
Current statusApr 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51338.png

Downloads & links

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Map

FileDownload / File: emd_51338.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFSC-calculated nominal resolution is 2.9 angstrom. Practical resolution on manual inspection is closer to 4 angstrom in some local areas. The DeepEMhancer map was generated and used for model building
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 299.52 Å		0.83 Å/pix.
x 360 pix.
= 299.52 Å		0.83 Å/pix.
x 360 pix.
= 299.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.6153495 - 1.0975705
Average (Standard dev.)0.0003948601 (±0.021454984)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 299.52002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51338_msk_1.map
Projections & Slices
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Half map: #2

Fileemd_51338_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_51338_half_map_2.map
Projections & Slices
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Sample components

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Entire : KBTBD4 WT-HDAC2 2:2 complex mediated by molecular glue UM171

EntireName: KBTBD4 WT-HDAC2 2:2 complex mediated by molecular glue UM171
Components
  • Complex: KBTBD4 WT-HDAC2 2:2 complex mediated by molecular glue UM171
    • Protein or peptide: Isoform 1 of Kelch repeat and BTB domain-containing protein 4
    • Protein or peptide: Histone deacetylase 2
  • Ligand: (1r,4r)-N~1~-[(7P)-2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine
  • Ligand: ZINC ION

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Supramolecule #1: KBTBD4 WT-HDAC2 2:2 complex mediated by molecular glue UM171

SupramoleculeName: KBTBD4 WT-HDAC2 2:2 complex mediated by molecular glue UM171
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Isoform 1 of Kelch repeat and BTB domain-containing protein 4

MacromoleculeName: Isoform 1 of Kelch repeat and BTB domain-containing protein 4
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.208742 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MESPEEPGAS MDENYFVNYT FKDRSHSGRV AQGIMKLCLE EELFADVTIS VEGREFQLHR LVLSAQSCFF RSMFTSNLKE AHNRVIVLQ DVSESVFQLL VDYIYHGTVK LRAEELQEIY EVSDMYQLTS LFEECSRFLA RTVQVGNCLQ VMWLADRHSD P ELYTAAKH ...String:
MESPEEPGAS MDENYFVNYT FKDRSHSGRV AQGIMKLCLE EELFADVTIS VEGREFQLHR LVLSAQSCFF RSMFTSNLKE AHNRVIVLQ DVSESVFQLL VDYIYHGTVK LRAEELQEIY EVSDMYQLTS LFEECSRFLA RTVQVGNCLQ VMWLADRHSD P ELYTAAKH CAKTHLAQLQ NTEEFLHLPH RLLTDIISDG VPCSQNPTEA IEAWINFNKE EREAFAESLR TSLKEIGENV HI YLIGKES SRTHSLAVSL HCAEDDSISV SGQNSLCHQI TAACKHGGDL YVVGGSIPRR MWKCNNATVD WEWCAPLPRD RLQ HTLVSV PGKDAIYSLG GKTLQDTLSN AVIYYRVGDN VWTETTQLEV AVSGAAGANL NGIIYLLGGE ENDLDFFTKP SRLI QCFDT ETDKCHVKPY VLPFAGRMHA AVHKDLVFIV AEGDSLVCYN PLLDSFTRLC LPEAWSSAPS LWKIASCNGS IYVFR DRYK KGDANTYKLD PATSAVTVTR GIKVLLTNLQ FVLA

UniProtKB: Kelch repeat and BTB domain-containing protein 4

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Macromolecule #2: Histone deacetylase 2

MacromoleculeName: Histone deacetylase 2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: histone deacetylase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.443156 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAYSQGGGKK KVCYYYDGDI GNYYYGQGHP MKPHRIRMTH NLLLNYGLYR KMEIYRPHKA TAEEMTKYHS DEYIKFLRSI RPDNMSEYS KQMQRFNVGE DCPVFDGLFE FCQLSTGGSV AGAVKLNRQQ TDMAVNWAGG LHHAKKSEAS GFCYVNDIVL A ILELLKYH ...String:
MAYSQGGGKK KVCYYYDGDI GNYYYGQGHP MKPHRIRMTH NLLLNYGLYR KMEIYRPHKA TAEEMTKYHS DEYIKFLRSI RPDNMSEYS KQMQRFNVGE DCPVFDGLFE FCQLSTGGSV AGAVKLNRQQ TDMAVNWAGG LHHAKKSEAS GFCYVNDIVL A ILELLKYH QRVLYIDIDI HHGDGVEEAF YTTDRVMTVS FHKYGEYFPG TGDLRDIGAG KGKYYAVNFP MRDGIDDESY GQ IFKPIIS KVMEMYQPSA VVLQCGADSL SGDRLGCFNL TVKGHAKCVE VVKTFNLPLL MLGGGGYTIR NVARCWTYET AVA LDCEIP NELPYNDYFE YFGPDFKLHI SPSNMTNQNT PEYMEKIKQR LFENLRMLPH APGVQMQAIP EDAVHEDSGD EDGE DPDKR ISIRASDKRI ACDEEFSDSE DEGEGGRRNV ADHKKGAKKA RIEEDKKETE DKKTDVKEED KSKDNSGEKT DTKGT KSEQ LSNP

UniProtKB: Histone deacetylase 2

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Macromolecule #3: (1r,4r)-N~1~-[(7P)-2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyri...

MacromoleculeName: (1r,4r)-N~1~-[(7P)-2-benzyl-7-(2-methyl-2H-tetrazol-5-yl)-9H-pyrimido[4,5-b]indol-4-yl]cyclohexane-1,4-diamine
type: ligand / ID: 3 / Number of copies: 2 / Formula: A1ACV
Molecular weightTheoretical: 453.542 Da

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium Chloride
5.0 mMMgCl2magnesium dichloride
5.0 %C3H5(OH)3glycerol
0.5 mMC9H15O6PTCEP
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 12732 / Average electron dose: 38.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 13588054
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 356933
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

7zzt

source_name: PDB, initial_model_type: experimental model

0-f1

source_name: AlphaFold, initial_model_type: in silico model
DetailsInitial local fitting was done using Chimera. Then Coot and Phenix was used for model refinement and validation.
RefinementProtocol: FLEXIBLE FIT / Overall B value: 122.7
Output model

PDB-9ggn:
Cryo-EM structure of KBTBD4 WT-HDAC2 2:2 complex mediated by molecular glue UM171

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