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- PDB-9sdx: Structure of RBR binding E2 variant crosslinked with NEDD8-CUL5-R... -

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Basic information

Entry
Database: PDB / ID: 9sdx
TitleStructure of RBR binding E2 variant crosslinked with NEDD8-CUL5-RBX2 bound ARIH2 and Ub
Components
  • Cullin-5
  • E3 ubiquitin-protein ligase ARIH2
  • L3A2-1
  • NEDD8
  • RING-box protein 2
  • Ubiquitin
KeywordsLIGASE / Complex
Function / homology
Function and homology information


negative regulation of focal adhesion disassembly / developmental cell growth / RBR-type E3 ubiquitin transferase / ERBB2 signaling pathway / negative regulation of focal adhesion assembly / reelin-mediated signaling pathway / cellular response to camptothecin / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / regulation of neuron migration ...negative regulation of focal adhesion disassembly / developmental cell growth / RBR-type E3 ubiquitin transferase / ERBB2 signaling pathway / negative regulation of focal adhesion assembly / reelin-mediated signaling pathway / cellular response to camptothecin / cullin-RING-type E3 NEDD8 transferase / NEDD8 transferase activity / regulation of neuron migration / protein K11-linked ubiquitination / protein neddylation / ubiquitin conjugating enzyme binding / NEDD8 ligase activity / response to redox state / : / Cul5-RING ubiquitin ligase complex / SCF ubiquitin ligase complex / hematopoietic stem cell proliferation / SCF-dependent proteasomal ubiquitin-dependent protein catabolic process / ubiquitin ligase complex scaffold activity / TGF-beta receptor signaling activates SMADs / regulation of proteolysis / regulation of postsynapse assembly / cullin family protein binding / anatomical structure morphogenesis / ubiquitin ligase complex / protein K63-linked ubiquitination / site of DNA damage / endoplasmic reticulum unfolded protein response / protein K48-linked ubiquitination / Maturation of protein E / Maturation of protein E / ER Quality Control Compartment (ERQC) / Myoclonic epilepsy of Lafora / FLT3 signaling by CBL mutants / IRAK2 mediated activation of TAK1 complex / Prevention of phagosomal-lysosomal fusion / Alpha-protein kinase 1 signaling pathway / Glycogen synthesis / IRAK1 recruits IKK complex / IRAK1 recruits IKK complex upon TLR7/8 or 9 stimulation / Endosomal Sorting Complex Required For Transport (ESCRT) / Membrane binding and targetting of GAG proteins / Negative regulation of FLT3 / Regulation of TBK1, IKKε (IKBKE)-mediated activation of IRF3, IRF7 / PTK6 Regulates RTKs and Their Effectors AKT1 and DOK1 / post-translational protein modification / Regulation of TBK1, IKKε-mediated activation of IRF3, IRF7 upon TLR3 ligation / IRAK2 mediated activation of TAK1 complex upon TLR7/8 or 9 stimulation / Constitutive Signaling by NOTCH1 HD Domain Mutants / NOTCH2 Activation and Transmission of Signal to the Nucleus / TICAM1,TRAF6-dependent induction of TAK1 complex / intrinsic apoptotic signaling pathway / TICAM1-dependent activation of IRF3/IRF7 / APC/C:Cdc20 mediated degradation of Cyclin B / Regulation of FZD by ubiquitination / Downregulation of ERBB4 signaling / APC-Cdc20 mediated degradation of Nek2A / p75NTR recruits signalling complexes / InlA-mediated entry of Listeria monocytogenes into host cells / TRAF6 mediated IRF7 activation in TLR7/8 or 9 signaling / NF-kB is activated and signals survival / TRAF6-mediated induction of TAK1 complex within TLR4 complex / Regulation of pyruvate metabolism / Pexophagy / Regulation of innate immune responses to cytosolic DNA / NRIF signals cell death from the nucleus / Downregulation of ERBB2:ERBB3 signaling / Regulation of PTEN localization / VLDLR internalisation and degradation / Activated NOTCH1 Transmits Signal to the Nucleus / Synthesis of active ubiquitin: roles of E1 and E2 enzymes / protein modification process / Translesion synthesis by REV1 / TICAM1, RIP1-mediated IKK complex recruitment / Regulation of BACH1 activity / Translesion synthesis by POLK / InlB-mediated entry of Listeria monocytogenes into host cell / JNK (c-Jun kinases) phosphorylation and activation mediated by activated human TAK1 / Activation of IRF3, IRF7 mediated by TBK1, IKKε (IKBKE) / MAP3K8 (TPL2)-dependent MAPK1/3 activation / Downregulation of TGF-beta receptor signaling / Translesion synthesis by POLI / Josephin domain DUBs / IKK complex recruitment mediated by RIP1 / Gap-filling DNA repair synthesis and ligation in GG-NER / PINK1-PRKN Mediated Mitophagy / TGF-beta receptor signaling in EMT (epithelial to mesenchymal transition) / Regulation of activated PAK-2p34 by proteasome mediated degradation / TNFR1-induced NF-kappa-B signaling pathway / TCF dependent signaling in response to WNT / Regulation of NF-kappa B signaling / activated TAK1 mediates p38 MAPK activation / Autodegradation of Cdh1 by Cdh1:APC/C / APC/C:Cdc20 mediated degradation of Securin / Asymmetric localization of PCP proteins / Regulation of signaling by CBL / N-glycan trimming in the ER and Calnexin/Calreticulin cycle / Ubiquitin-dependent degradation of Cyclin D
Similarity search - Function
: / : / E3 ubiquitin-protein ligase ARIH2, N-terminal UBA domain / Ariadne domain / Ariadne domain / E3 ubiquitin ligase RBR family / IBR domain, a half RING-finger domain / Nedd8-like ubiquitin / IBR domain / IBR domain ...: / : / E3 ubiquitin-protein ligase ARIH2, N-terminal UBA domain / Ariadne domain / Ariadne domain / E3 ubiquitin ligase RBR family / IBR domain, a half RING-finger domain / Nedd8-like ubiquitin / IBR domain / IBR domain / In Between Ring fingers / TRIAD supradomain / TRIAD supradomain profile. / Zinc finger, RING-H2-type / RING-H2 zinc finger domain / Cullin, N-terminal / Cullin protein neddylation domain / : / Cullin, conserved site / Cullin alpha solenoid domain / Cullin family signature. / Cullin repeat-like-containing domain superfamily / Cullin protein, neddylation domain / Cullin / Cullin protein neddylation domain / Cullin / : / Cullin alpha+beta domain / Cullin homology domain / Cullin homology domain superfamily / Cullin family profile. / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / : / Ubiquitin domain signature. / Ubiquitin conserved site / Ubiquitin domain / 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
5-azanylpentan-2-one / E3 ubiquitin-protein ligase ARIH2 / Polyubiquitin-C / Ubiquitin-like protein NEDD8 / Cullin-5 / RING-box protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsSchulman, B.A. / Du, J.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)SCHU3196/1-1 Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2026
Title: E2 variants for probing E3 ubiquitin ligase activities.
Authors: Jiale Du / Gisele A Andree / Daniel Horn-Ghetko / Luca Stier / Jaspal Singh / Sebastian Kostrhon / Leo Kiss / Matthias Mann / Sachdev S Sidhu / Brenda A Schulman /
Abstract: E3 ligases partner with E2 enzymes to regulate vast eukaryotic biology. The hierarchical nature of these pairings, with >600 E3s and ~40 E2s in humans, necessitates that E2s cofunction with numerous ...E3 ligases partner with E2 enzymes to regulate vast eukaryotic biology. The hierarchical nature of these pairings, with >600 E3s and ~40 E2s in humans, necessitates that E2s cofunction with numerous different E3s. Here, focusing on E3s in the RING-between-RING (RBR) family and their partner UBE2L3 and UBE2D-family E2s, we report an approach to interrogate selected pathways. We screened phage-displayed libraries of structure-based E2 variants (E2Vs) to discover enzymes with enhanced affinity and specificity toward half of all RBR E3 ligases (ARIH1, ARIH2, ANKIB1, CUL9, HOIL1, HOIP, and RNF14). Collectively, these E2Vs allowed distinguishing actions of different cofunctioning E3s, obtaining high-resolution cryogenic Electron Microscopy (cryo-EM) structures of an RBR E3 in the context of a substrate-bound multiprotein complex, and profiling an endogenous RBR E3 response to an extracellular stimulus. Overall, we anticipate that E2V technology will be a generalizable tool to enable in-depth mechanistic and structural analysis of E3 ligase functions, and mapping their activity states and protein partners in cellular signaling cascades.
History
DepositionAug 15, 2025Deposition site: PDBE / Processing site: PDBE
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
N: NEDD8
R: RING-box protein 2
U: Ubiquitin
G: L3A2-1
C: Cullin-5
H: E3 ubiquitin-protein ligase ARIH2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,84114
Polymers197,2826
Non-polymers5598
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 NRUGCH

#1: 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: 9086.562 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
#2: Protein RING-box protein 2 / Rbx2 / CKII beta-binding protein 1 / CKBBP1 / RING finger protein 7 / Regulator of cullins 2 / ...Rbx2 / CKII beta-binding protein 1 / CKBBP1 / RING finger protein 7 / Regulator of cullins 2 / Sensitive to apoptosis gene protein


Mass: 12697.436 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RNF7, RBX2, ROC2, SAG / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q9UBF6, RING-type E3 ubiquitin transferase, cullin-RING-type E3 NEDD8 transferase
#3: Protein Ubiquitin


Mass: 8519.778 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBC / Production host: Escherichia coli (E. coli) / References: UniProt: P0CG48
#4: Protein L3A2-1


Mass: 17998.607 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
#5: Protein Cullin-5 / CUL-5 / Vasopressin-activated calcium-mobilizing receptor 1 / VACM-1


Mass: 91085.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CUL5, VACM1 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: Q93034
#6: Protein E3 ubiquitin-protein ligase ARIH2 / ARI-2 / Protein ariadne-2 homolog / RING-type E3 ubiquitin transferase ARIH2 / Triad1 protein


Mass: 57893.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ARIH2, ARI2, TRIAD1, HT005 / Production host: Escherichia coli (E. coli)
References: UniProt: O95376, RBR-type E3 ubiquitin transferase

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Non-polymers , 2 types, 8 molecules

#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-SY8 / 5-azanylpentan-2-one


Mass: 101.147 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H11NO / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
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

ComponentName: NEDD8-CUL5-RBX2-ARIH2~E2V~Ub / Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightValue: 200 kDa/nm / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.1_5286model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 200442 / Symmetry type: POINT
RefinementHighest resolution: 2.97 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210762
ELECTRON MICROSCOPYf_angle_d0.58514590
ELECTRON MICROSCOPYf_dihedral_angle_d4.4231474
ELECTRON MICROSCOPYf_chiral_restr0.041668
ELECTRON MICROSCOPYf_plane_restr0.0041872

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