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- PDB-8rx0: (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G76S, K48C)-UBE2R1(C93K, S138C, C1... -

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Entry
Database: PDB / ID: 8rx0
Title(NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G76S, K48C)-UBE2R1(C93K, S138C, C191S, C223S)-Ub
Components
  • Cullin-2
  • E3 ubiquitin-protein ligase RBX1, N-terminally processed
  • Elongin-B
  • Elongin-C
  • Isoform B of Bromodomain-containing protein 4
  • NEDD8
  • Polyubiquitin-B
  • Ubiquitin-conjugating enzyme E2 R1
  • von Hippel-Lindau disease tumor suppressor
KeywordsLIGASE / ubiquitin / cullin / RING / E2 complex
Function / homology
Function and homology information


regulation of cellular response to hypoxia / negative regulation of beige fat cell differentiation / negative regulation of receptor signaling pathway via JAK-STAT / RHOBTB3 ATPase cycle / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / (E3-independent) E2 ubiquitin-conjugating enzyme / cullin-RING ubiquitin ligase complex / Cul7-RING ubiquitin ligase complex / cellular response to chemical stress ...regulation of cellular response to hypoxia / negative regulation of beige fat cell differentiation / negative regulation of receptor signaling pathway via JAK-STAT / RHOBTB3 ATPase cycle / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / (E3-independent) E2 ubiquitin-conjugating enzyme / cullin-RING ubiquitin ligase complex / Cul7-RING ubiquitin ligase complex / cellular response to chemical stress / transcription elongation factor activity / target-directed miRNA degradation / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / elongin complex / protein K27-linked ubiquitination / positive regulation of protein autoubiquitination / RNA polymerase II transcription initiation surveillance / protein neddylation / hypothalamus gonadotrophin-releasing hormone neuron development / Replication of the SARS-CoV-1 genome / female meiosis I / NEDD8 ligase activity / positive regulation of protein monoubiquitination / VCB complex / fat pad development / negative regulation of response to oxidative stress / mitochondrion transport along microtubule / Cul5-RING ubiquitin ligase complex / intracellular membraneless organelle / ubiquitin-ubiquitin ligase activity / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / SCF ubiquitin ligase complex / E2 ubiquitin-conjugating enzyme / Cul2-RING ubiquitin ligase complex / negative regulation of type I interferon production / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / female gonad development / SUMOylation of ubiquitinylation proteins / seminiferous tubule development / negative regulation of mitophagy / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / Prolactin receptor signaling / histone H4K8ac reader activity / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / histone H3K9ac reader activity / RNA polymerase II C-terminal domain binding / histone H3K27ac reader activity / ubiquitin conjugating enzyme activity / TGF-beta receptor signaling activates SMADs / P-TEFb complex binding / male meiosis I / negative regulation of DNA damage checkpoint / histone H4 reader activity / negative regulation of transcription elongation by RNA polymerase II / Pausing and recovery of Tat-mediated HIV elongation / Tat-mediated HIV elongation arrest and recovery / regulation of proteolysis / histone H4K5ac reader activity / cullin family protein binding / histone H4K12ac reader activity / HIV elongation arrest and recovery / Pausing and recovery of HIV elongation / host-mediated suppression of viral transcription / histone H4K16ac reader activity / regulation of postsynapse assembly / positive regulation of intrinsic apoptotic signaling pathway by p53 class mediator / positive regulation of G2/M transition of mitotic cell cycle / DNA replication initiation / protein monoubiquitination / anatomical structure morphogenesis / negative regulation of signal transduction / positive regulation of T-helper 17 cell lineage commitment / Tat-mediated elongation of the HIV-1 transcript / site of DNA damage / Formation of HIV-1 elongation complex containing HIV-1 Tat / Formation of HIV elongation complex in the absence of HIV Tat / protein K48-linked ubiquitination / ubiquitin-like ligase-substrate adaptor activity / energy homeostasis / RNA Polymerase II Transcription Elongation / neuron projection morphogenesis / cellular response to interferon-beta / Formation of RNA Pol II elongation complex / signal transduction in response to DNA damage / Nuclear events stimulated by ALK signaling in cancer / regulation of proteasomal protein catabolic process / negative regulation of TORC1 signaling / transcription-coupled nucleotide-excision repair / Maturation of protein E / Maturation of protein E / RNA Polymerase II Pre-transcription Events / positive regulation of TORC1 signaling / regulation of cellular response to insulin stimulus / ER Quality Control Compartment (ERQC) / RNA polymerase II CTD heptapeptide repeat kinase activity / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants
Similarity search - Function
Nedd8-like ubiquitin / von Hippel-Lindau disease tumour suppressor, beta/alpha domain / von Hippel-Lindau disease tumour suppressor, alpha domain / von Hippel-Lindau disease tumour suppressor, beta domain / VHL superfamily / von Hippel-Lindau disease tumour suppressor, alpha domain superfamily / von Hippel-Lindau disease tumour suppressor, beta domain superfamily / VHL beta domain / VHL box domain / Zinc finger, RING-H2-type ...Nedd8-like ubiquitin / von Hippel-Lindau disease tumour suppressor, beta/alpha domain / von Hippel-Lindau disease tumour suppressor, alpha domain / von Hippel-Lindau disease tumour suppressor, beta domain / VHL superfamily / von Hippel-Lindau disease tumour suppressor, alpha domain superfamily / von Hippel-Lindau disease tumour suppressor, beta domain superfamily / VHL beta domain / VHL box domain / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / : / Cullin protein neddylation domain / : / Cullin, conserved site / Elongin-C / Cullin family signature. / Elongin B / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin, N-terminal / Cullin alpha solenoid domain / Cullin / Cullin homology domain / Cullin homology domain superfamily / Cullin family profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Ubiquitin-conjugating enzyme, active site / Ubiquitin-conjugating (UBC) active site signature. / Bromodomain protein 4, C-terminal / C-terminal domain of bromodomain protein 4 / Ubiquitin-conjugating enzyme E2 / Ubiquitin-conjugating enzyme / Ubiquitin-conjugating (UBC) core domain profile. / Ubiquitin-conjugating enzyme E2, catalytic domain homologues / Ubiquitin-conjugating enzyme/RWD-like / Brdt, bromodomain, repeat I / Brdt, bromodomain, repeat II / NET domain superfamily / NET domain profile. / : / NET domain / Bromodomain extra-terminal - transcription regulation / SKP1/BTB/POZ domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Bromodomain, conserved site / Bromodomain signature. / Bromodomain / bromo domain / Bromodomain / Bromodomain (BrD) profile. / Bromodomain-like superfamily / Ubiquitin family / Ubiquitin homologues / Ubiquitin domain profile. / Ubiquitin-like domain / Zinc finger, RING/FYVE/PHD-type / Ubiquitin-like domain superfamily / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Chem-759 / Bromodomain-containing protein 4 / Polyubiquitin-B / von Hippel-Lindau disease tumor suppressor / Ubiquitin-conjugating enzyme E2 R1 / E3 ubiquitin-protein ligase RBX1 / Cullin-2 / Elongin-C / Elongin-B / Ubiquitin-like protein NEDD8
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsCrowe, C. / Nakasone, M.A. / Ciulli, A.
Funding supportEuropean Union, Switzerland, United Kingdom, 5items
OrganizationGrant numberCountry
European Research Council (ERC)ERC-2012-StG-311460European Union
Innovative Medicines Initiative875510 Switzerland
Wellcome Trust223816/Z/21/Z United Kingdom
Medical Research Council (MRC, United Kingdom)4050845509 United Kingdom
European Research Council (ERC)851478European Union
Citation
Journal: Sci Adv / Year: 2024
Title: Mechanism of degrader-targeted protein ubiquitinability.
Authors: Charlotte Crowe / Mark A Nakasone / Sarah Chandler / Conner Craigon / Gajanan Sathe / Michael H Tatham / Nikolai Makukhin / Ronald T Hay / Alessio Ciulli /
Abstract: Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived ...Small-molecule degraders of disease-driving proteins offer a clinically proven modality with enhanced therapeutic efficacy and potential to tackle previously undrugged targets. Stable and long-lived degrader-mediated ternary complexes drive fast and profound target degradation; however, the mechanisms by which they affect target ubiquitination remain elusive. Here, we show cryo-EM structures of the VHL Cullin 2 RING E3 ligase with the degrader MZ1 directing target protein Brd4 toward UBE2R1-ubiquitin, and Lys at optimal positioning for nucleophilic attack. In vitro ubiquitination and mass spectrometry illuminate a patch of favorably ubiquitinable lysines on one face of Brd4, with cellular degradation and ubiquitinomics confirming the importance of Lys and nearby Lys/Lys, identifying the "ubiquitination zone." Our results demonstrate the proficiency of MZ1 in positioning the substrate for catalysis, the favorability of Brd4 for ubiquitination by UBE2R1, and the flexibility of CRL2 for capturing suboptimal lysines. We propose a model for ubiquitinability of degrader-recruited targets, providing a mechanistic blueprint for further rational drug design.
#1: Journal: Biorxiv / Year: 2024
Title: Mechanism of degrader-targeted protein ubiquitinability
Authors: Crowe, C. / Nakasone, M.A. / Chandler, S. / Tatham, M.H. / Makukhin, N. / Hay, R.T. / Ciulli, A.
History
DepositionFeb 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 2.0Sep 4, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Experimental preparation / Non-polymer description / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / audit_author ...atom_site / audit_author / chem_comp / chem_comp_atom / chem_comp_bond / citation_author / em_entity_assembly / em_imaging / em_sample_support / em_software / em_vitrification / entity / entity_name_com / entity_poly / entity_poly_seq / entity_src_gen / pdbx_contact_author / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_struct_oper_list / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_close_contact / pdbx_validate_main_chain_plane / pdbx_validate_peptide_omega / pdbx_validate_planes / pdbx_validate_polymer_linkage / pdbx_validate_rmsd_angle / pdbx_validate_rmsd_bond / pdbx_validate_torsion / struct_asym / struct_conf / struct_conn / struct_mon_prot_cis / struct_ref / struct_ref_seq / struct_ref_seq_dif / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _chem_comp.formula / _chem_comp.formula_weight ..._chem_comp.formula / _chem_comp.formula_weight / _chem_comp.id / _chem_comp.mon_nstd_flag / _chem_comp.name / _chem_comp.type / _em_entity_assembly.entity_id_list / _em_imaging.c2_aperture_diameter / _em_vitrification.instrument / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_poly.pdbx_strand_id / _entity_src_gen.gene_src_common_name / _entity_src_gen.pdbx_end_seq_num / _entity_src_gen.pdbx_gene_src_gene / _pdbx_struct_assembly.details / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_oper_list.symmetry_operation / _struct_ref.db_code / _struct_ref.db_name / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_db_isoform / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_auth_seq_align_end / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq.pdbx_strand_id / _struct_ref_seq.seq_align_beg / _struct_ref_seq.seq_align_end / _struct_sheet.number_strands
Description: Atomic clashes / Provider: author / Type: Coordinate replacement
Revision 2.1Oct 23, 2024Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details / Item: _pdbx_entry_details.has_protein_modification
Revision 3.0Dec 24, 2025Group: Data collection / Non-polymer description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / entity
Item: _chem_comp.formula / _chem_comp.formula_weight / _entity.formula_weight

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform B of Bromodomain-containing protein 4
B: von Hippel-Lindau disease tumor suppressor
D: Elongin-C
C: Elongin-B
E: Cullin-2
G: Ubiquitin-conjugating enzyme E2 R1
N: NEDD8
R: E3 ubiquitin-protein ligase RBX1, N-terminally processed
U: Polyubiquitin-B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,62313
Polymers196,4249
Non-polymers1,1994
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area20420 Å2
ΔGint-141 kcal/mol
Surface area77760 Å2

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Components

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Protein , 9 types, 9 molecules ABDCEGNRU

#1: Protein Isoform B of Bromodomain-containing protein 4 / Protein HUNK1


Mass: 12978.103 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
#2: Protein von Hippel-Lindau disease tumor suppressor / Protein G7 / pVHL


Mass: 18558.162 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: VHL / Production host: Escherichia coli (E. coli) / References: UniProt: P40337
#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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELOC, TCEB1 / Production host: Escherichia coli (E. coli) / References: UniProt: Q15369
#4: 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.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ELOB, TCEB2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q15370
#5: Protein Cullin-2 / CUL-2


Mass: 87098.930 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q13617
#6: Protein Ubiquitin-conjugating enzyme E2 R1 / (E3-independent) E2 ubiquitin-conjugating enzyme R1 / E2 ubiquitin-conjugating enzyme R1 / ...(E3-independent) E2 ubiquitin-conjugating enzyme R1 / E2 ubiquitin-conjugating enzyme R1 / Ubiquitin-conjugating enzyme E2-32 kDa complementing / Ubiquitin-conjugating enzyme E2-CDC34 / Ubiquitin-protein ligase R1


Mass: 26762.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CDC34, UBCH3, UBE2R1 / Production host: Escherichia coli (E. coli)
References: UniProt: P49427, E2 ubiquitin-conjugating enzyme, (E3-independent) E2 ubiquitin-conjugating enzyme
#7: Protein NEDD8 / Neddylin / Neural precursor cell expressed developmentally down-regulated protein 8 / NEDD-8 / ...Neddylin / Neural precursor cell expressed developmentally down-regulated protein 8 / NEDD-8 / Ubiquitin-like protein Nedd8


Mass: 8661.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NEDD8 / Production host: Escherichia coli (E. coli) / References: UniProt: Q15843
#8: Protein E3 ubiquitin-protein ligase RBX1, N-terminally processed / E3 ubiquitin-protein transferase RBX1 / N-terminally processed


Mass: 11196.830 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RBX1, RNF75, ROC1 / Production host: Escherichia coli (E. coli) / References: UniProt: P62877
#9: Protein Polyubiquitin-B


Mass: 8576.831 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBB / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG47

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Non-polymers , 2 types, 4 molecules

#10: Chemical ChemComp-759 / (2~{S},4~{R})-1-[(2~{S})-2-[2-[2-[2-[2-[2-[(9~{S})-7-(4-chlorophenyl)-4,5,13-trimethyl-3-thia-1,8,11,12-tetrazatricyclo[8.3.0.0^{2,6}]trideca-2(6),4,7,10,12-pentaen-9-yl]ethanoylamino]ethoxy]ethoxy]ethoxy]ethanoylamino]-3,3-dimethyl-butanoyl]-~{N}-[[4-(4-methyl-2,3-dihydro-1,3-thiazol-5-yl)phenyl]methyl]-4-oxidanyl-pyrrolidine-2-carboxamide


Mass: 1002.639 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C49H60ClN9O8S2 / Feature type: SUBJECT OF INVESTIGATION
#11: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn

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Details

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: (NEDD8)-CRL2VHL-MZ1-Brd4BD2-Ub(G76S, K48C)-UBE2R1(C93K, S138C, C191S, C223S)-Ub
Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Molecular weightValue: 0.19 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK IV / 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 defocus max: 3000 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 38 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149823 / Symmetry type: POINT

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