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- EMDB-40477: KLHDC2 in complex with EloB and EloC -

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Basic information

Entry
Database: EMDB / ID: EMD-40477
TitleKLHDC2 in complex with EloB and EloC
Map data
Sample
  • Complex: Full-length KLHDC2 in complex with EloB and EloC
    • Protein or peptide: Kelch domain-containing protein 2
    • Protein or peptide: Elongin-B
    • Protein or peptide: Elongin-C
KeywordsKLHDC2 / PEPTIDE BINDING PROTEIN
Function / homology
Function and homology information


ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / target-directed miRNA degradation / elongin complex / VCB complex / Cul5-RING ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation ...ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / target-directed miRNA degradation / elongin complex / VCB complex / Cul5-RING ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / ubiquitin-like ligase-substrate adaptor activity / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / Formation of HIV elongation complex in the absence of HIV Tat / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / RNA Polymerase II Pre-transcription Events / transcription corepressor binding / transcription elongation by RNA polymerase II / TP53 Regulates Transcription of DNA Repair Genes / transcription initiation at RNA polymerase II promoter / Vif-mediated degradation of APOBEC3G / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Inactivation of CSF3 (G-CSF) signaling / Evasion by RSV of host interferon responses / Regulation of expression of SLITs and ROBOs / Antigen processing: Ubiquitination & Proteasome degradation / positive regulation of proteasomal ubiquitin-dependent protein catabolic process / protein-macromolecule adaptor activity / Neddylation / ubiquitin-dependent protein catabolic process / protein-containing complex assembly / nuclear membrane / proteasome-mediated ubiquitin-dependent protein catabolic process / nuclear body / protein ubiquitination / ubiquitin protein ligase binding / regulation of transcription by RNA polymerase II / nucleoplasm / nucleus / cytosol
Similarity search - Function
Galactose oxidase, central domain / Elongin B / Elongin-C / Kelch-type beta propeller / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / SKP1/BTB/POZ domain superfamily / Ubiquitin family ...Galactose oxidase, central domain / Elongin B / Elongin-C / Kelch-type beta propeller / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / SKP1/BTB/POZ domain superfamily / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Ubiquitin-like domain superfamily
Similarity search - Domain/homology
Elongin-C / Elongin-B / Kelch domain-containing protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.74 Å
AuthorsDigianantonio KM / Bekes M
Funding support United States, 1 items
OrganizationGrant numberCountry
Other private United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Co-opting the E3 ligase KLHDC2 for targeted protein degradation by small molecules.
Authors: Christopher M Hickey / Katherine M Digianantonio / Kurt Zimmermann / Alicia Harbin / Connor Quinn / Avani Patel / Peter Gareiss / Amanda Chapman / Bernadette Tiberi / Jennifer Dobrodziej / ...Authors: Christopher M Hickey / Katherine M Digianantonio / Kurt Zimmermann / Alicia Harbin / Connor Quinn / Avani Patel / Peter Gareiss / Amanda Chapman / Bernadette Tiberi / Jennifer Dobrodziej / John Corradi / Angela M Cacace / David R Langley / Miklós Békés /
Abstract: Targeted protein degradation (TPD) by PROTAC (proteolysis-targeting chimera) and molecular glue small molecules is an emerging therapeutic strategy. To expand the roster of E3 ligases that can be ...Targeted protein degradation (TPD) by PROTAC (proteolysis-targeting chimera) and molecular glue small molecules is an emerging therapeutic strategy. To expand the roster of E3 ligases that can be utilized for TPD, we describe the discovery and biochemical characterization of small-molecule ligands targeting the E3 ligase KLHDC2. Furthermore, we functionalize these KLHDC2-targeting ligands into KLHDC2-based BET-family and AR PROTAC degraders and demonstrate KLHDC2-dependent target-protein degradation. Additionally, we offer insight into the assembly of the KLHDC2 E3 ligase complex. Using biochemical binding studies, X-ray crystallography and cryo-EM, we show that the KLHDC2 E3 ligase assembles into a dynamic tetramer held together via its own C terminus, and that this assembly can be modulated by substrate and ligand engagement.
History
DepositionApr 13, 2023-
Header (metadata) releaseJan 3, 2024-
Map releaseJan 3, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40477.png

Downloads & links

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Map

FileDownload / File: emd_40477.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.0493
Minimum - Maximum-0.76971555 - 1.0691468
Average (Standard dev.)0.0012418639 (±0.015256168)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40477_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_40477_additional_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #2

Fileemd_40477_half_map_1.map
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Histogram

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Half map: #1

Fileemd_40477_half_map_2.map
Projections & Slices
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Sample components

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Entire : Full-length KLHDC2 in complex with EloB and EloC

EntireName: Full-length KLHDC2 in complex with EloB and EloC
Components
  • Complex: Full-length KLHDC2 in complex with EloB and EloC
    • Protein or peptide: Kelch domain-containing protein 2
    • Protein or peptide: Elongin-B
    • Protein or peptide: Elongin-C

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Supramolecule #1: Full-length KLHDC2 in complex with EloB and EloC

SupramoleculeName: Full-length KLHDC2 in complex with EloB and EloC / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Kelch domain-containing protein 2

MacromoleculeName: Kelch domain-containing protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.909402 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GHHHHHHADG NEDLRADDLP GPAFESYESM ELACPAERSG HVAVSDGRHM FVWGGYKSNQ VRGLYDFYLP REELWIYNME TGRWKKINT EGDVPPSMSG SCAVCVDRVL YLFGGHHSRG NTNKFYMLDS RSTDRVLQWE RIDCQGIPPS SKDKLGVWVY K NKLIFFGG ...String:
GHHHHHHADG NEDLRADDLP GPAFESYESM ELACPAERSG HVAVSDGRHM FVWGGYKSNQ VRGLYDFYLP REELWIYNME TGRWKKINT EGDVPPSMSG SCAVCVDRVL YLFGGHHSRG NTNKFYMLDS RSTDRVLQWE RIDCQGIPPS SKDKLGVWVY K NKLIFFGG YGYLPEDKVL GTFEFDETSF WNSSHPRGWN DHVHILDTET FTWSQPITTG KAPSPRAAHA CATVGNRGFV FG GRYRDAR MNDLHYLNLD TWEWNELIPQ GICPVGRSWH SLTPVSSDHL FLFGGFTTDK QPLSDAWTYC ISKNEWIQFN HPY TEKPRL WHTACASDEG EVIVFGGCAN NLLVHHRAAH SNEILIFSVQ PKSLVRLSLE AVICFKEMLA NSWNCLPKHL LHSV NQRFG SNNTSGS

UniProtKB: Kelch domain-containing protein 2

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Macromolecule #2: Elongin-B

MacromoleculeName: Elongin-B / type: protein_or_peptide / ID: 2 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.748406 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MDVFLMIRRH KTTIFTDAKE SSTVFELKRI VEGILKRPPD EQRLYKDDQL LDDGKTLGEC GFTSQTARPQ APATVGLAFR ADDTFEALC IEPFSSPPEL PDVMK

UniProtKB: Elongin-B

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Macromolecule #3: Elongin-C

MacromoleculeName: Elongin-C / type: protein_or_peptide / ID: 3 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.84342 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
MYVKLISSDG HEFIVKREHA LTSGTIKAML SGPGQFAENE TNEVNFREIP SHVLSKVCMY FTYKVRYTNS STEIPEFPIA PEIALELLM AANFLDC

UniProtKB: Elongin-C

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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

Concentration5 mg/mL
BufferpH: 7.3
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
1.0 mMC9H15O6PTCEP
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
Details: SPT Labtech Quantifoil Active Grid nanowire grids were used (1.2uM/0.8uM). The grids were glow-discharged with the in-line glow discharge to activate the nanowires at 12mA. Approximately 6 ...Details: SPT Labtech Quantifoil Active Grid nanowire grids were used (1.2uM/0.8uM). The grids were glow-discharged with the in-line glow discharge to activate the nanowires at 12mA. Approximately 6 nL of the complex were applied to the grids using a Chameleon EP system (SPT Labtech) at ambient temperature with 75% relative humidity.
VitrificationCryogen name: ETHANE / Instrument: SPOTITON
DetailsThis sample was crosslinked with BS3 prior to freezing

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 2 / Number real images: 10382 / Average electron dose: 58.1 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4153896
Startup modelType of model: OTHER
Details: ab-initio reconstruction with 6 classes, and one class demonstrated good density for 4 KLHDC2 Kelch domains (cryoSPARC 3.3.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.1) / Number images used: 185914
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Details: homogeneous refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Details: non-uniform refinement
Final 3D classificationNumber classes: 20 / Software - Name: cryoSPARC (ver. 4.1.1) / Details: 3DVA analyzed in cluster mode with 20 clusters
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

5t35

chain_id: B, source_name: PDB, initial_model_type: experimental model

5t35

chain_id: C, source_name: PDB, initial_model_type: experimental model

l_v4

source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 337
Output model

PDB-8sh2:
KLHDC2 in complex with EloB and EloC

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