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

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Basic information

Entry
Database: PDB / ID: 8sh2
TitleKLHDC2 in complex with EloB and EloC
Components
  • Elongin-B
  • Elongin-C
  • Kelch domain-containing protein 2
KeywordsPEPTIDE BINDING PROTEIN / KLHDC2
Function / homology
Function and homology information


ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / target-directed miRNA degradation / VCB complex / elongin 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 / VCB complex / elongin 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 / Tat-mediated elongation of the HIV-1 transcript / ubiquitin-like ligase-substrate adaptor activity / 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 / TP53 Regulates Transcription of DNA Repair Genes / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / Vif-mediated degradation of APOBEC3G / Inactivation of CSF3 (G-CSF) signaling / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / 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 / Neddylation / ubiquitin-dependent protein catabolic process / protein-containing complex assembly / nuclear membrane / protein-macromolecule adaptor activity / 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-C / Elongin B / 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-C / Elongin B / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.74 Å
AuthorsDigianantonio, K.M. / Bekes, M.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 3, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Feb 28, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Kelch domain-containing protein 2
E: Elongin-B
F: Elongin-C
G: Elongin-B
H: Elongin-C
I: Elongin-B
J: Elongin-C
K: Elongin-B
L: Elongin-C
C: Kelch domain-containing protein 2
A: Kelch domain-containing protein 2
D: Kelch domain-containing protein 2


Theoretical massNumber of molelcules
Total (without water)278,00512
Polymers278,00512
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Kelch domain-containing protein 2 / Hepatocellular carcinoma-associated antigen 33 / Host cell factor homolog LCP / Host cell factor- ...Hepatocellular carcinoma-associated antigen 33 / Host cell factor homolog LCP / Host cell factor-like protein 1 / HCLP-1


Mass: 46909.402 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: KLHDC2, HCA33 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9Y2U9
#2: Protein
Elongin-B / EloB / Elongin 18 kDa subunit / RNA polymerase II transcription factor SIII subunit B / SIII p18 / ...EloB / Elongin 18 kDa subunit / RNA polymerase II transcription factor SIII subunit B / SIII p18 / Transcription elongation factor B polypeptide 2


Mass: 11748.406 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELOB, TCEB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q15370
#3: Protein
Elongin-C / EloC / Elongin 15 kDa subunit / RNA polymerase II transcription factor SIII subunit C / SIII p15 / ...EloC / Elongin 15 kDa subunit / RNA polymerase II transcription factor SIII subunit C / SIII p15 / Transcription elongation factor B polypeptide 1


Mass: 10843.420 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELOC, TCEB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q15369

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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: Full-length KLHDC2 in complex with EloB and EloC / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.3
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaCl1
31 mMTCEPC9H15O6P1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample was crosslinked with BS3 prior to freezing
Specimen supportDetails: 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.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: SPOTITON / Cryogen name: ETHANE

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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: 2500 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 58.1 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 10382
EM imaging opticsEnergyfilter name: GIF Quantum LS

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
4cryoSPARC3.3.1CTF correction
7PHENIX1.20.1model fitting
9cryoSPARC3.3.1initial Euler assignment
10cryoSPARC3.3.1final Euler assignment
11cryoSPARC4.1.1classification
12cryoSPARC4.1.13D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4153896
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.74 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 185914 / Symmetry type: POINT
Atomic model buildingB value: 337 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDSource nameType
15T35

5t35

B5T35B1PDBexperimental model
25T35

5t35

C5T35C1PDBexperimental model
3KLHDC2_AF-Q9Y2U9-F1-model_V42AlphaFoldin silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00718540
ELECTRON MICROSCOPYf_angle_d1.30125144
ELECTRON MICROSCOPYf_dihedral_angle_d7.7022437
ELECTRON MICROSCOPYf_chiral_restr0.072669
ELECTRON MICROSCOPYf_plane_restr0.0093243

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