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- PDB-8q7r: Ubiquitin ligation to substrate by a cullin-RING E3 ligase & Cdc3... -

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Basic information

Entry
Database: PDB / ID: 8q7r
TitleUbiquitin ligation to substrate by a cullin-RING E3 ligase & Cdc34: NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2~donor UB-Sil1 peptide
Components
  • Cullin-2
  • E3 ubiquitin-protein ligase RBX1
  • Elongin-B
  • Elongin-C
  • Nucleotide exchange factor SIL1
  • Protein fem-1 homolog C
  • Ubiquitin
  • Ubiquitin-conjugating enzyme E2 R2
KeywordsLIGASE / CUL2 / FEM1C / ELOBC / SIL1 / Ubiquitin / Ubiquitin Ligase / Ubiquitylation / monoubiquitylation / Elongin B / Elongin C
Function / homology
Function and homology information


adenyl-nucleotide exchange factor activity / cotranslational protein targeting to membrane / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / target-directed miRNA degradation / VCB complex ...adenyl-nucleotide exchange factor activity / cotranslational protein targeting to membrane / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / cullin-RING ubiquitin ligase complex / cellular response to chemical stress / Cul7-RING ubiquitin ligase complex / ubiquitin-dependent protein catabolic process via the C-end degron rule pathway / target-directed miRNA degradation / VCB complex / elongin complex / Loss of Function of FBXW7 in Cancer and NOTCH1 Signaling / positive regulation of protein autoubiquitination / protein neddylation / NEDD8 ligase activity / Cul5-RING ubiquitin ligase complex / negative regulation of response to oxidative stress / SCF ubiquitin ligase complex / Cul2-RING ubiquitin ligase complex / Cul4A-RING E3 ubiquitin ligase complex / ubiquitin-ubiquitin ligase activity / E2 ubiquitin-conjugating enzyme / negative regulation of type I interferon production / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / Cul3-RING ubiquitin ligase complex / Cul4B-RING E3 ubiquitin ligase complex / ubiquitin ligase complex scaffold activity / Prolactin receptor signaling / ubiquitin conjugating enzyme activity / cullin family protein binding / 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 / protein monoubiquitination / ubiquitin ligase complex / 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 / protein K48-linked ubiquitination / RNA Polymerase II Transcription Elongation / Nuclear events stimulated by ALK signaling in cancer / Formation of RNA Pol II elongation complex / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / RNA Polymerase II Pre-transcription Events / Prevention of phagosomal-lysosomal fusion / IRAK2 mediated activation of TAK1 complex / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Membrane binding and targetting of GAG proteins / Endosomal Sorting Complex Required For Transport (ESCRT) / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / positive regulation of TORC1 signaling / Negative regulation of FLT3 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / Constitutive Signaling by NOTCH1 HD Domain Mutants / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / regulation of cellular response to insulin stimulus / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / TICAM1-dependent activation of IRF3/IRF7 / Regulation of FZD by ubiquitination / APC/C:Cdc20 mediated degradation of Cyclin B / Downregulation of ERBB4 signaling / p75NTR recruits signalling complexes / APC-Cdc20 mediated degradation of Nek2A / Regulation of innate immune responses to cytosolic DNA / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / NF-kB is activated and signals survival / post-translational protein modification / Downregulation of ERBB2:ERBB3 signaling / Pexophagy / intrinsic apoptotic signaling pathway / NRIF signals cell death from the nucleus / VLDLR internalisation and degradation / Regulation of PTEN localization / Activated NOTCH1 Transmits Signal to the Nucleus / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / Regulation of BACH1 activity / TICAM1, RIP1-mediated IKK complex recruitment / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Translesion synthesis by REV1 / Translesion synthesis by POLK / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / Downregulation of TGF-beta receptor signaling / InlB-mediated entry of Listeria monocytogenes into host cell / Regulation of activated PAK-2p34 by proteasome mediated degradation / Josephin domain DUBs / Translesion synthesis by POLI
Similarity search - Function
Nucleotide exchange factor Fes1 / : / Nucleotide exchange factor Fes1 / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / : / Cullin protein neddylation domain / : / Elongin-C / Elongin B ...Nucleotide exchange factor Fes1 / : / Nucleotide exchange factor Fes1 / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / : / Cullin protein neddylation domain / : / Elongin-C / Elongin B / Cullin, conserved site / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin / Cullin, N-terminal / Cullin homology domain / Cullin homology domain superfamily / Cullin family / 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. / 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 / Ankyrin repeat / SKP1/BTB/POZ domain superfamily / Ankyrin repeats (3 copies) / Ankyrin repeat profile. / Ankyrin repeat region circular profile. / ankyrin repeats / Ankyrin repeat / Ankyrin repeat-containing domain superfamily / Zinc finger RING-type profile. / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / Armadillo-like helical / Ubiquitin family / Ubiquitin homologues / Tetratricopeptide-like helical domain superfamily / Ubiquitin domain profile. / Ubiquitin-like domain / Armadillo-type fold / 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
: / Polyubiquitin-C / E3 ubiquitin-protein ligase RBX1 / Cullin-2 / Elongin-C / Elongin-B / Ubiquitin-conjugating enzyme E2 R2 / Protein fem-1 homolog C / Nucleotide exchange factor SIL1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsLiwocha, J. / Prabu, J.R. / Kleiger, G. / Schulman, B.A.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Mol Cell / Year: 2024
Title: Cullin-RING ligases employ geometrically optimized catalytic partners for substrate targeting.
Authors: Jerry Li / Nicholas Purser / Joanna Liwocha / Daniel C Scott / Holly A Byers / Barbara Steigenberger / Spencer Hill / Ishita Tripathi-Giesgen / Trent Hinkle / Fynn M Hansen / J Rajan Prabu / ...Authors: Jerry Li / Nicholas Purser / Joanna Liwocha / Daniel C Scott / Holly A Byers / Barbara Steigenberger / Spencer Hill / Ishita Tripathi-Giesgen / Trent Hinkle / Fynn M Hansen / J Rajan Prabu / Senthil K Radhakrishnan / Donald S Kirkpatrick / Kurt M Reichermeier / Brenda A Schulman / Gary Kleiger /
Abstract: Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. ...Cullin-RING ligases (CRLs) ubiquitylate specific substrates selected from other cellular proteins. Substrate discrimination and ubiquitin transferase activity were thought to be strictly separated. Substrates are recognized by substrate receptors, such as Fbox or BCbox proteins. Meanwhile, CRLs employ assorted ubiquitin-carrying enzymes (UCEs, which are a collection of E2 and ARIH-family E3s) specialized for either initial substrate ubiquitylation (priming) or forging poly-ubiquitin chains. We discovered specific human CRL-UCE pairings governing substrate priming. The results reveal pairing of CUL2-based CRLs and UBE2R-family UCEs in cells, essential for efficient PROTAC-induced neo-substrate degradation. Despite UBE2R2's intrinsic programming to catalyze poly-ubiquitylation, CUL2 employs this UCE for geometrically precise PROTAC-dependent ubiquitylation of a neo-substrate and for rapid priming of substrates recruited to diverse receptors. Cryo-EM structures illuminate how CUL2-based CRLs engage UBE2R2 to activate substrate ubiquitylation. Thus, pairing with a specific UCE overcomes E2 catalytic limitations to drive substrate ubiquitylation and targeted protein degradation.
History
DepositionAug 16, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 21, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 17, 2024Group: Derived calculations / Category: struct_sheet / struct_sheet_range
Revision 1.3Apr 24, 2024Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.4Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _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: Cullin-2
H: Protein fem-1 homolog C
U: Ubiquitin
F: Nucleotide exchange factor SIL1
C: Ubiquitin-conjugating enzyme E2 R2
D: Elongin-C
G: Elongin-B
R: E3 ubiquitin-protein ligase RBX1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)300,66712
Polymers300,3548
Non-polymers3134
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 7 types, 7 molecules AHUCDGR

#1: 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: Trichoplusia ni (cabbage looper) / References: UniProt: Q13617
#2: Protein Protein fem-1 homolog C / FEM1c / FEM1-gamma


Mass: 68767.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FEM1C, KIAA1785 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q96JP0
#3: Protein Ubiquitin


Mass: 77146.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: ...Details: MQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGMQIFVKTLTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLHLVLRLRGGV
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P0CG48
#5: Protein Ubiquitin-conjugating enzyme E2 R2 / E2 ubiquitin-conjugating enzyme R2 / Ubiquitin carrier protein R2 / Ubiquitin-conjugating enzyme E2- ...E2 ubiquitin-conjugating enzyme R2 / Ubiquitin carrier protein R2 / Ubiquitin-conjugating enzyme E2-CDC34B / Ubiquitin-protein ligase R2


Mass: 27190.932 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBE2R2, CDC34B, UBC3B / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: Q712K3, E2 ubiquitin-conjugating enzyme
#6: 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: 12485.135 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 BL21 (bacteria) / References: UniProt: Q15369
#7: 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: 13147.781 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 BL21 (bacteria) / References: UniProt: Q15370
#8: Protein E3 ubiquitin-protein ligase RBX1 / E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / ...E3 ubiquitin-protein transferase RBX1 / Protein ZYP / RING finger protein 75 / RING-box protein 1 / Rbx1 / Regulator of cullins 1 / ROC1


Mass: 12289.977 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: Trichoplusia ni (cabbage looper)
References: UniProt: P62877, RING-type E3 ubiquitin transferase, cullin-RING-type E3 NEDD8 transferase

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Protein/peptide , 1 types, 1 molecules F

#4: Protein/peptide Nucleotide exchange factor SIL1 / BiP-associated protein / BAP


Mass: 2227.457 Da / Num. of mol.: 1 / Mutation: D442C S455P L456T L457Q K458G E459R L460A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SIL1, UNQ545/PRO836 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: Q9H173

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Non-polymers , 2 types, 4 molecules

#9: Chemical ChemComp-U9O / 5-azanyl-1-oxidanyl-pentan-2-one


Mass: 117.146 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H11NO2
#10: 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 interestN
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Ubiquitin ligation to substrate by a cullin-RING E3 ligase & Cdc34: NEDD8-CUL2-RBX1-ELOB/C-FEM1C with trapped UBE2R2~donor UB-Sil1 peptideCOMPLEX#1-#80MULTIPLE SOURCES
2RING E3 ligase (RBX1) and Cullin-2 (CUL2)COMPLEX#1, #81RECOMBINANT
3CDC34, NEDD8, ELOB, ELOC, FEM1C with UBE2R2-donor UB-Sil1 peptideCOMPLEX#2-#71RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Trichoplusia ni (cabbage looper)7111
33Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 600 nm
Image recordingElectron dose: 66 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: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135564 / Symmetry type: POINT

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