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- PDB-9cht: Human E3 ligase E6AP in complex with HPV16-E6 and p53 -

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Basic information

Entry
Database: PDB / ID: 9cht
TitleHuman E3 ligase E6AP in complex with HPV16-E6 and p53
Components
  • Immunoglobulin G-binding protein G/Cellular tumor antigen p53 fusion protein
  • Protein E6
  • Ubiquitin-protein ligase E3A
KeywordsLIGASE / Complex / Viral / Ubiquitination
Function / homology
Function and homology information


sperm entry / positive regulation of Golgi lumen acidification / symbiont-mediated suppression of host transcription / negative regulation of dendritic spine morphogenesis / motor learning / regulation of ubiquitin-dependent protein catabolic process / symbiont-mediated perturbation of host apoptosis / prostate gland growth / HECT-type E3 ubiquitin transferase / activation of GTPase activity ...sperm entry / positive regulation of Golgi lumen acidification / symbiont-mediated suppression of host transcription / negative regulation of dendritic spine morphogenesis / motor learning / regulation of ubiquitin-dependent protein catabolic process / symbiont-mediated perturbation of host apoptosis / prostate gland growth / HECT-type E3 ubiquitin transferase / activation of GTPase activity / Loss of function of TP53 in cancer due to loss of tetramerization ability / Regulation of TP53 Expression / negative regulation of helicase activity / signal transduction by p53 class mediator / negative regulation of G1 to G0 transition / negative regulation of glucose catabolic process to lactate via pyruvate / Transcriptional activation of cell cycle inhibitor p21 / regulation of intrinsic apoptotic signaling pathway by p53 class mediator / negative regulation of pentose-phosphate shunt / ATP-dependent DNA/DNA annealing activity / Activation of NOXA and translocation to mitochondria / regulation of cell cycle G2/M phase transition / regulation of fibroblast apoptotic process / intrinsic apoptotic signaling pathway in response to hypoxia / oligodendrocyte apoptotic process / negative regulation of miRNA processing / positive regulation of thymocyte apoptotic process / oxidative stress-induced premature senescence / regulation of tissue remodeling / glucose catabolic process to lactate via pyruvate / positive regulation of mitochondrial membrane permeability / positive regulation of programmed necrotic cell death / mRNA transcription / bone marrow development / circadian behavior / regulation of mitochondrial membrane permeability involved in apoptotic process / germ cell nucleus / RUNX3 regulates CDKN1A transcription / IgG binding / TP53 regulates transcription of additional cell cycle genes whose exact role in the p53 pathway remain uncertain / TP53 Regulates Transcription of Death Receptors and Ligands / Activation of PUMA and translocation to mitochondria / regulation of DNA damage response, signal transduction by p53 class mediator / histone deacetylase regulator activity / negative regulation of glial cell proliferation / Regulation of TP53 Activity through Association with Co-factors / negative regulation of neuroblast proliferation / T cell lineage commitment / mitochondrial DNA repair / Formation of Senescence-Associated Heterochromatin Foci (SAHF) / ER overload response / B cell lineage commitment / thymocyte apoptotic process / TP53 Regulates Transcription of Caspase Activators and Caspases / negative regulation of mitophagy / cardiac septum morphogenesis / negative regulation of DNA replication / entrainment of circadian clock by photoperiod / PI5P Regulates TP53 Acetylation / negative regulation of telomere maintenance via telomerase / Zygotic genome activation (ZGA) / positive regulation of release of cytochrome c from mitochondria / Association of TriC/CCT with target proteins during biosynthesis / necroptotic process / locomotory exploration behavior / TP53 Regulates Transcription of Genes Involved in Cytochrome C Release / rRNA transcription / TFIID-class transcription factor complex binding / SUMOylation of transcription factors / TP53 regulates transcription of several additional cell death genes whose specific roles in p53-dependent apoptosis remain uncertain / intrinsic apoptotic signaling pathway by p53 class mediator / androgen receptor signaling pathway / T cell proliferation involved in immune response / negative regulation of reactive oxygen species metabolic process / positive regulation of execution phase of apoptosis / Transcriptional Regulation by VENTX / regulation of proteolysis / postsynaptic cytosol / replicative senescence / cellular response to UV-C / general transcription initiation factor binding / intrinsic apoptotic signaling pathway in response to endoplasmic reticulum stress / cellular response to actinomycin D / neuroblast proliferation / positive regulation of RNA polymerase II transcription preinitiation complex assembly / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / response to X-ray / type II interferon-mediated signaling pathway / hematopoietic stem cell differentiation / Pyroptosis / chromosome organization / viral process / embryonic organ development / somitogenesis / TP53 Regulates Transcription of Genes Involved in G1 Cell Cycle Arrest / glial cell proliferation / hematopoietic progenitor cell differentiation / protein K48-linked ubiquitination / progesterone receptor signaling pathway / core promoter sequence-specific DNA binding
Similarity search - Function
Ubiquitin-protein ligase E3A / Ubiquitin-protein ligase E3A, N-terminal zinc-binding domain / Ubiquitin-protein ligase E3A, N-terminal zinc-binding domain superfamily / Amino-terminal Zinc-binding domain of ubiquitin ligase E3A / Ubiquitin-protein ligase E3B/C / E6 early regulatory protein / E6 superfamily / Early Protein (E6) / IgG-binding B / B domain ...Ubiquitin-protein ligase E3A / Ubiquitin-protein ligase E3A, N-terminal zinc-binding domain / Ubiquitin-protein ligase E3A, N-terminal zinc-binding domain superfamily / Amino-terminal Zinc-binding domain of ubiquitin ligase E3A / Ubiquitin-protein ligase E3B/C / E6 early regulatory protein / E6 superfamily / Early Protein (E6) / IgG-binding B / B domain / M protein-type anchor domain / GA-like domain / GA-like domain / HECT domain / HECT, E3 ligase catalytic domain / HECT-domain (ubiquitin-transferase) / HECT domain profile. / Domain Homologous to E6-AP Carboxyl Terminus with / Immunoglobulin/albumin-binding domain superfamily / Cellular tumor antigen p53, transactivation domain 2 / Transactivation domain 2 / p53 transactivation domain / P53 transactivation motif / p53 family signature. / p53, tetramerisation domain / P53 tetramerisation motif / p53, DNA-binding domain / P53 DNA-binding domain / p53 tumour suppressor family / p53-like tetramerisation domain superfamily / p53/RUNT-type transcription factor, DNA-binding domain superfamily / YSIRK Gram-positive signal peptide / LPXTG cell wall anchor motif / Gram-positive cocci surface proteins LPxTG motif profile. / LPXTG cell wall anchor domain / p53-like transcription factor, DNA-binding
Similarity search - Domain/homology
Protein E6 / Cellular tumor antigen p53 / Immunoglobulin G-binding protein G / Ubiquitin-protein ligase E3A
Similarity search - Component
Biological speciesHomo sapiens (human)
Streptococcus sp. group G (bacteria)
Human papillomavirus 16
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsKenny, S. / Das, C.
Funding support United States, 3items
OrganizationGrant numberCountry
Other privateP30CA023168
American Heart Association905924/SK/2021 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM132024 United States
CitationJournal: Structure / Year: 2025
Title: Structure of E6AP in complex with HPV16-E6 and p53 reveals a novel ordered domain important for E3 ligase activation.
Authors: Sebastian Kenny / Shalini Iyer / Clinton A Gabel / Natalia Tegenfeldt / Andrew G DeMarco / Mark C Hall / Leifu Chang / V Jo Davisson / Scott Vande Pol / Chittaranjan Das /
Abstract: High-risk human papillomavirus E6 oncoprotein is a model system for the recognition and degradation of cellular p53 tumor suppressor protein. There remains a gap in the understanding of the ubiquitin ...High-risk human papillomavirus E6 oncoprotein is a model system for the recognition and degradation of cellular p53 tumor suppressor protein. There remains a gap in the understanding of the ubiquitin transfer reaction, including placement of the E6AP catalytic HECT domain of the ligase concerning the p53 substrate and how E6 itself is protected from ubiquitination. We determined the cryoelectron microscopy (cryo-EM) structure of the E6AP/E6/p53 complex, related the structure to in vivo modeling of the tri-molecular complex, and identified structural interactions associated with activation of the ubiquitin ligase function. The structure reveals that the N-terminal ordered domain (NOD) in E6AP has a terminal alpha helix that mediates the interaction of the NOD with the HECT domain of E6AP and protects the HPV-E6 protein from ubiquitination. In addition, this NOD helix is required for E6AP ligase function by contributing to the affinity of the E6-E6AP association, modulating E6 substrate recognition, while displacing UbcH7.
History
DepositionJul 2, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 8, 2025Provider: repository / Type: Initial release
Revision 1.1Jan 29, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Mar 19, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Remark 650HELIX DETERMINATION METHOD: AUTHOR
Remark 700SHEET DETERMINATION METHOD: AUTHOR

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ubiquitin-protein ligase E3A
B: Immunoglobulin G-binding protein G/Cellular tumor antigen p53 fusion protein
C: Protein E6


Theoretical massNumber of molelcules
Total (without water)165,1503
Polymers165,1503
Non-polymers00
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

#1: Protein Ubiquitin-protein ligase E3A / E6AP ubiquitin-protein ligase / HECT-type ubiquitin transferase E3A / Human papillomavirus E6- ...E6AP ubiquitin-protein ligase / HECT-type ubiquitin transferase E3A / Human papillomavirus E6-associated protein / Oncogenic protein-associated protein E6-AP / Renal carcinoma antigen NY-REN-54


Mass: 102982.867 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: UBE3A, E6AP, EPVE6AP, HPVE6A / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q05086, HECT-type E3 ubiquitin transferase
#2: Antibody Immunoglobulin G-binding protein G/Cellular tumor antigen p53 fusion protein


Mass: 43801.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus sp. group G (bacteria), (gene. exp.) Homo sapiens (human)
Gene: spg, TP53, P53 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P06654, UniProt: P04637
#3: Protein Protein E6


Mass: 18365.369 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human papillomavirus 16 / Gene: E6 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03126
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: Ternary complex of E6AP with viral factor E6 and neosubstrate p53
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3)
Buffer solutionpH: 7.4
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse, further purified by SEC
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 279 K

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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: 1500 nm / Nominal defocus min: 600 nm
Image recordingAverage exposure time: 3.192 sec. / Electron dose: 1.52 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5548

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.2.0particle selection
4cryoSPARC3.2.0CTF correction
7Coot0.8.9.2model fitting
9cryoSPARC3.2.0initial Euler assignment
10cryoSPARC3.2.0final Euler assignment
11cryoSPARC3.2.0classification
12cryoSPARC3.2.03D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1591147
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 238679 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Atomic model buildingSource name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0038624
ELECTRON MICROSCOPYf_angle_d0.64311649
ELECTRON MICROSCOPYf_dihedral_angle_d4.6571158
ELECTRON MICROSCOPYf_chiral_restr0.0431270
ELECTRON MICROSCOPYf_plane_restr0.0051514

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