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- PDB-8rwz: Open non-crosslinked structure Brd4BD2-MZ1-(NEDD8)-CRL2VHL -

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Basic information

Entry
Database: PDB / ID: 8rwz
TitleOpen non-crosslinked structure Brd4BD2-MZ1-(NEDD8)-CRL2VHL
Components
  • Bromodomain-containing protein 4
  • Cullin-2
  • E3 ubiquitin-protein ligase RBX1, N-terminally processed
  • Elongin-B
  • Elongin-C
  • von Hippel-Lindau disease tumor suppressor
KeywordsLIGASE / BET bromodomain / E3 ligase / PROTAC
Function / homology
Function and homology information


regulation of cellular response to hypoxia / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / negative regulation of receptor signaling pathway via JAK-STAT / RHOBTB3 ATPase cycle / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / cellular response to chemical stress / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling ...regulation of cellular response to hypoxia / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / negative regulation of receptor signaling pathway via JAK-STAT / RHOBTB3 ATPase cycle / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / cellular response to chemical stress / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / transcription elongation factor activity / target-directed miRNA degradation / elongin complex / positive regulation of protein autoubiquitination / RNA polymerase II transcription initiation surveillance / protein neddylation / Replication of the SARS-CoV-1 genome / NEDD8 ligase activity / negative regulation of response to oxidative stress / VCB complex / Cul5-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / negative regulation of type I interferon production / ubiquitin-ubiquitin ligase activity / intracellular membraneless organelle / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul2-RING ubiquitin ligase complex / Cul3-RING ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / Cul4-RING E3 ubiquitin ligase complex / SUMOylation of ubiquitinylation proteins / negative regulation of mitophagy / Prolactin receptor signaling / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / RNA polymerase II C-terminal domain binding / P-TEFb complex binding / negative regulation of DNA damage checkpoint / histone H4 reader activity / cullin family protein binding / negative regulation of transcription elongation by RNA polymerase II / 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 / host-mediated suppression of viral transcription / protein monoubiquitination / positive regulation of G2/M transition of mitotic cell cycle / negative regulation of signal transduction / positive regulation of T-helper 17 cell lineage commitment / Tat-mediated elongation of the HIV-1 transcript / Formation of HIV-1 elongation complex containing HIV-1 Tat / ubiquitin-like ligase-substrate adaptor activity / protein K48-linked ubiquitination / Formation of HIV elongation complex in the absence of HIV Tat / Nuclear events stimulated by ALK signaling in cancer / RNA Polymerase II Transcription Elongation / Formation of RNA Pol II elongation complex / : / negative regulation of TORC1 signaling / transcription-coupled nucleotide-excision repair / RNA Polymerase II Pre-transcription Events / positive regulation of TORC1 signaling / regulation of cellular response to insulin stimulus / RNA polymerase II CTD heptapeptide repeat kinase activity / negative regulation of insulin receptor signaling pathway / intrinsic apoptotic signaling pathway / protein serine/threonine kinase binding / negative regulation of autophagy / post-translational protein modification / T cell activation / Regulation of BACH1 activity / transcription corepressor binding / condensed nuclear chromosome / transcription coregulator activity / TP53 Regulates Transcription of DNA Repair Genes / cellular response to amino acid stimulus / positive regulation of cell differentiation / transcription initiation at RNA polymerase II promoter / Degradation of DVL / transcription elongation by RNA polymerase II / positive regulation of transcription elongation by RNA polymerase II / Degradation of GLI1 by the proteasome / GSK3B and BTRC:CUL1-mediated-degradation of NFE2L2 / Negative regulation of NOTCH4 signaling / G1/S transition of mitotic cell cycle / Recognition of DNA damage by PCNA-containing replication complex / negative regulation of canonical Wnt signaling pathway / Hedgehog 'on' state / Vif-mediated degradation of APOBEC3G / FBXL7 down-regulates AURKA during mitotic entry and in early mitosis / Degradation of GLI2 by the proteasome / GLI3 is processed to GLI3R by the proteasome / RING-type E3 ubiquitin transferase / Inactivation of CSF3 (G-CSF) signaling / Degradation of beta-catenin by the destruction complex / DNA Damage Recognition in GG-NER / Oxygen-dependent proline hydroxylation of Hypoxia-inducible Factor Alpha / Evasion by RSV of host interferon responses / NOTCH1 Intracellular Domain Regulates Transcription
Similarity search - Function
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 ...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 / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Elongin-C / Elongin B / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin alpha solenoid domain / Cullin family profile. / S-phase kinase-associated protein 1-like / SKP1 component, POZ domain / Skp1 family, tetramerisation domain / Found in Skp1 protein family / Bromodomain protein 4, C-terminal / C-terminal domain of bromodomain protein 4 / 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 / 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 / Elongin-C / Bromodomain-containing protein 4 / von Hippel-Lindau disease tumor suppressor / E3 ubiquitin-protein ligase RBX1 / Cullin-2 / Elongin-B
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsCrowe, C. / Nakasone, M.A. / Ciulli, A.
Funding supportEuropean Union, United Kingdom, Switzerland, 4items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
Medical Research Council (MRC, United Kingdom) United Kingdom
Wellcome Trust United Kingdom
Innovative Medicines Initiative Switzerland
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 2.0Oct 2, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Polymer sequence / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / audit_author ...atom_site / audit_author / em_software / entity / entity_name_com / entity_poly / entity_poly_seq / entity_src_gen / pdbx_contact_author / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_conn_angle / 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 / refine_ls_restr / struct_conf / struct_conn / struct_conn_type / struct_mon_prot_cis / struct_ref / struct_ref_seq / struct_ref_seq_dif / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _audit_author.name / _entity.formula_weight ..._audit_author.name / _entity.formula_weight / _entity.pdbx_description / _entity_poly.pdbx_seq_one_letter_code / _entity_poly.pdbx_seq_one_letter_code_can / _entity_src_gen.gene_src_common_name / _entity_src_gen.pdbx_end_seq_num / _entity_src_gen.pdbx_gene_src_gene / _pdbx_contact_author.id / _pdbx_nonpoly_scheme.auth_seq_num / _struct_mon_prot_cis.pdbx_omega_angle / _struct_ref.db_code / _struct_ref.db_name / _struct_ref.pdbx_align_begin / _struct_ref.pdbx_db_accession / _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.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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bromodomain-containing protein 4
B: von Hippel-Lindau disease tumor suppressor
C: Elongin-B
D: Elongin-C
E: Cullin-2
R: E3 ubiquitin-protein ligase RBX1, N-terminally processed
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,99910
Polymers152,7986
Non-polymers1,2014
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

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Protein , 6 types, 6 molecules ABCDER

#1: Protein Bromodomain-containing protein 4 / Protein HUNK1


Mass: 13281.371 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-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
#4: Protein Elongin-C


Mass: 10914.498 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / References: UniProt: L7N190
#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 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

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Non-polymers , 2 types, 4 molecules

#7: 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: 1004.655 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C49H62ClN9O8S2 / Feature type: SUBJECT OF INVESTIGATION
#8: 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: Open non-crosslinked structure Brd4BD2-MZ1-(NEDD8)-CRL2VHL
Type: COMPLEX / Entity ID: #1-#6 / 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
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3200 nm / Nominal defocus min: 1700 nm
Image recordingElectron dose: 26 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 132697 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0024637
ELECTRON MICROSCOPYf_angle_d0.5495781
ELECTRON MICROSCOPYf_dihedral_angle_d7.0891154
ELECTRON MICROSCOPYf_chiral_restr00
ELECTRON MICROSCOPYf_plane_restr0.0031154

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