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- PDB-9i2c: Cryo-EM structure of KBTBD4 WT-HDAC2-CoREST1 2:1:1 complex mediat... -

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Basic information

Entry
Database: PDB / ID: 9i2c
TitleCryo-EM structure of KBTBD4 WT-HDAC2-CoREST1 2:1:1 complex mediated by molecular glue UM171
Components
  • Histone deacetylase 2
  • Isoform 1 of Kelch repeat and BTB domain-containing protein 4
  • REST corepressor 1
KeywordsLIGASE / E3 ligase / deacetylase / ubiquitylation / transcription / molecular glue / complex
Function / homology
Function and homology information


positive regulation of male mating behavior / protein de-2-hydroxyisobutyrylase activity / protein lysine delactylase activity / p75NTR negatively regulates cell cycle via SC1 / fungiform papilla formation / epidermal cell differentiation / eyelid development in camera-type eye / negative regulation of dendritic spine development / positive regulation of megakaryocyte differentiation / histone decrotonylase activity ...positive regulation of male mating behavior / protein de-2-hydroxyisobutyrylase activity / protein lysine delactylase activity / p75NTR negatively regulates cell cycle via SC1 / fungiform papilla formation / epidermal cell differentiation / eyelid development in camera-type eye / negative regulation of dendritic spine development / positive regulation of megakaryocyte differentiation / histone decrotonylase activity / NuRD complex / positive regulation of interleukin-1 production / DNA repair complex / : / regulation of cell fate specification / EGR2 and SOX10-mediated initiation of Schwann cell myelination / negative regulation of stem cell population maintenance / regulation of stem cell differentiation / histone deacetylase activity, hydrolytic mechanism / histone deacetylase / ESC/E(Z) complex / cardiac muscle hypertrophy / positive regulation of intracellular estrogen receptor signaling pathway / behavioral response to ethanol / odontogenesis of dentin-containing tooth / STAT3 nuclear events downstream of ALK signaling / cellular response to dopamine / embryonic digit morphogenesis / histone deacetylase activity / protein lysine deacetylase activity / Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In linear amides / response to caffeine / Notch-HLH transcription pathway / positive regulation of oligodendrocyte differentiation / Sin3-type complex / dendrite development / positive regulation of stem cell population maintenance / transcription repressor complex / histone deacetylase complex / response to amyloid-beta / histone methyltransferase complex / progesterone receptor signaling pathway / positive regulation of proteolysis / RNA Polymerase I Transcription Initiation / response to hyperoxia / hair follicle placode formation / cellular response to transforming growth factor beta stimulus / Regulation of MECP2 expression and activity / FOXO-mediated transcription of oxidative stress, metabolic and neuronal genes / positive regulation of epithelial to mesenchymal transition / NF-kappaB binding / Transcriptional regulation of brown and beige adipocyte differentiation by EBF2 / cellular response to retinoic acid / Regulation of TP53 Activity through Acetylation / MECP2 regulates neuronal receptors and channels / heat shock protein binding / response to amphetamine / negative regulation of cell migration / regulation of embryonic development / erythrocyte differentiation / negative regulation of transforming growth factor beta receptor signaling pathway / Regulation of PTEN gene transcription / transcription coregulator binding / SUMOylation of chromatin organization proteins / ERCC6 (CSB) and EHMT2 (G9a) positively regulate rRNA expression / response to nicotine / circadian regulation of gene expression / response to cocaine / Regulation of endogenous retroelements by KRAB-ZFP proteins / negative regulation of neuron projection development / HDACs deacetylate histones / promoter-specific chromatin binding / Regulation of endogenous retroelements by Piwi-interacting RNAs (piRNAs) / Negative Regulation of CDH1 Gene Transcription / NoRC negatively regulates rRNA expression / cellular response to hydrogen peroxide / NOTCH1 Intracellular Domain Regulates Transcription / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / histone deacetylase binding / positive regulation of tumor necrosis factor production / transcription corepressor activity / cellular response to heat / transcription regulator complex / Factors involved in megakaryocyte development and platelet production / response to lipopolysaccharide / heterochromatin formation / histone binding / chromatin organization / Potential therapeutics for SARS / proteasome-mediated ubiquitin-dependent protein catabolic process / RNA polymerase II-specific DNA-binding transcription factor binding / chromosome, telomeric region / response to xenobiotic stimulus / chromatin remodeling / negative regulation of gene expression / negative regulation of DNA-templated transcription / positive regulation of cell population proliferation / chromatin binding / regulation of transcription by RNA polymerase II
Similarity search - Function
Kelch repeat and BTB domain-containing protein 4 / KBTBD4, BTB/POZ domain / KBTBD4, BACK domain / Kelch repeat type 2 / Kelch motif / : / Helical region in REST corepressor / : / ELM2 domain / ELM2 domain ...Kelch repeat and BTB domain-containing protein 4 / KBTBD4, BTB/POZ domain / KBTBD4, BACK domain / Kelch repeat type 2 / Kelch motif / : / Helical region in REST corepressor / : / ELM2 domain / ELM2 domain / ELM2 domain profile. / ELM2 / Histone deacetylase / BTB-kelch protein / BTB/Kelch-associated / BTB And C-terminal Kelch / BTB And C-terminal Kelch / SANT domain profile. / SANT domain / Histone deacetylase family / Kelch-type beta propeller / Histone deacetylase domain / Histone deacetylase domain superfamily / Histone deacetylase domain / Myb-like DNA-binding domain / Ureohydrolase domain superfamily / BTB/POZ domain / BTB domain profile. / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / Broad-Complex, Tramtrack and Bric a brac / BTB/POZ domain / SKP1/BTB/POZ domain superfamily / Homeobox-like domain superfamily
Similarity search - Domain/homology
: / Histone deacetylase 2 / Kelch repeat and BTB domain-containing protein 4 / REST corepressor 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsChen, Z. / Chi, G. / Pike, A.C.W. / Montes, B. / Bullock, A.N.
Funding support United Kingdom, Switzerland, 2items
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
DepositionJan 20, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 9, 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: Isoform 1 of Kelch repeat and BTB domain-containing protein 4
B: Histone deacetylase 2
C: Isoform 1 of Kelch repeat and BTB domain-containing protein 4
D: REST corepressor 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,6116
Polymers194,0924
Non-polymers5192
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Isoform 1 of Kelch repeat and BTB domain-containing protein 4 / BTB and kelch domain-containing protein 4


Mass: 58208.742 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KBTBD4, BKLHD4 / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q9NVX7
#2: Protein Histone deacetylase 2 / HD2 / Protein deacylase HDAC2


Mass: 55443.156 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HDAC2 / Cell line (production host): Expi293F / Production host: Homo sapiens (human)
References: UniProt: Q92769, histone deacetylase, Hydrolases; Acting on carbon-nitrogen bonds, other than peptide bonds; In linear amides
#3: Protein REST corepressor 1 / Protein CoREST


Mass: 22231.699 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RCOR1, KIAA0071, RCOR / Cell line (production host): Expi293F / Production host: Homo sapiens (human) / References: UniProt: Q9UKL0
#4: Chemical ChemComp-A1ACV / (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


Mass: 453.542 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C25H27N9 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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: KBTBD4 WT-HDAC2-CoREST1 2:1:1 complex mediated by molecular glue UM171
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
35 mMmagnesium dichlorideMgCl21
45 %glycerolC3H5(OH)31
50.5 mMTCEPC9H15O6P1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1000 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 38 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 12732
Image scansWidth: 5760 / Height: 4092

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 13588054
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105484 / Symmetry type: POINT
Atomic model buildingB value: 85.1 / Protocol: FLEXIBLE FIT
Details: Initial local fitting was done using Chimera. Then Coot and Phenix were used for model refinement and validation.
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
17ZZT

7zzt

17ZZT1PDBexperimental model
21AF-Q9N010-F12AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00110127
ELECTRON MICROSCOPYf_angle_d0.38513844
ELECTRON MICROSCOPYf_dihedral_angle_d9.4193241
ELECTRON MICROSCOPYf_chiral_restr0.0391637
ELECTRON MICROSCOPYf_plane_restr0.0031763

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