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- PDB-8dlf: EBNA1 DNA binding domain (DBD) (458-617)+2 repeats of family repe... -

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Basic information

Entry
Database: PDB / ID: 8dlf
TitleEBNA1 DNA binding domain (DBD) (458-617)+2 repeats of family repeat (FR) region
Components
  • (2XFR DNA (56-MER)) x 2
  • Epstein-Barr nuclear antigen 1
KeywordsVIRAL PROTEIN/DNA / EBNA1 / OriP / EBV / family repeats (FR) / VIRAL PROTEIN-DNA complex
Function / homology
Function and homology information


host cell PML body / symbiont-mediated suppression of host antigen processing and presentation / viral latency / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / enzyme-substrate adaptor activity / symbiont-mediated disruption of host cell PML body / regulation of DNA replication / endonuclease activity / DNA-binding transcription factor activity / virus-mediated perturbation of host defense response ...host cell PML body / symbiont-mediated suppression of host antigen processing and presentation / viral latency / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / enzyme-substrate adaptor activity / symbiont-mediated disruption of host cell PML body / regulation of DNA replication / endonuclease activity / DNA-binding transcription factor activity / virus-mediated perturbation of host defense response / positive regulation of DNA-templated transcription / DNA binding
Similarity search - Function
Epstein Barr virus nuclear antigen-1, DNA-binding / Epstein Barr virus nuclear antigen-1, DNA-binding domain / Epstein Barr virus nuclear antigen-1, DNA-binding domain superfamily / E2/EBNA1, C-terminal
Similarity search - Domain/homology
DNA / DNA (> 10) / Epstein-Barr nuclear antigen 1
Similarity search - Component
Biological speciesHuman herpesvirus 4 strain B95-8 (Epstein-Barr virus (strain B95-8))
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsMei, Y. / Lieberman, P.M. / Murakami, K.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)RO1 CA093606 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)RO1 CA259171 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1-GM123233 United States
National Science Foundation (NSF, United States)MCB 2131806 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30 CA0101815 United States
CitationJournal: J Virol / Year: 2022
Title: Cryo-EM Structure and Functional Studies of EBNA1 Binding to the Family of Repeats and Dyad Symmetry Elements of Epstein-Barr Virus .
Authors: Yang Mei / Troy E Messick / Jayaraju Dheekollu / Hee Jong Kim / Sudheer Molugu / Leonardo Josué Castro Muñoz / Vera Moiskeenkova-Bell / Kenji Murakami / Paul M Lieberman /
Abstract: Epstein-Barr nuclear antigen 1 (EBNA1) is a multifunctional viral-encoded DNA-binding protein essential for Epstein-Barr virus (EBV) DNA replication and episome maintenance. EBNA1 binds to two ...Epstein-Barr nuclear antigen 1 (EBNA1) is a multifunctional viral-encoded DNA-binding protein essential for Epstein-Barr virus (EBV) DNA replication and episome maintenance. EBNA1 binds to two functionally distinct elements at the viral origin of plasmid replication (), termed the dyad symmetry (DS) element, required for replication initiation and the family of repeats (FR) required for episome maintenance. Here, we determined the cryo-electron microscopy (cryo-EM) structure of the EBNA1 DNA binding domain (DBD) from amino acids (aa) 459 to 614 and its interaction with two tandem sites at the DS and FR. We found that EBNA1 induces a strong DNA bending angle in the DS, while the FR is more linear. The N-terminal arm of the DBD (aa 444 to 468) makes extensive contact with DNA as it wraps around the minor groove, with some conformational variation among EBNA1 monomers. Mutation of variable-contact residues K460 and K461 had only minor effects on DNA binding but had abrogated -dependent DNA replication. We also observed that the AT-rich intervening DNA between EBNA1 binding sites in the FR can be occupied by the EBNA1 AT hook, N-terminal domain (NTD) aa 1 to 90 to form a Zn-dependent stable complex with EBNA1 DBD on a 2×FR DNA template. We propose a model showing EBNA1 DBD and NTD cobinding at the FR and suggest that this may contribute to the oligomerization of viral episomes important for maintenance during latent infection. EBV latent infection is causally linked to diverse cancers and autoimmune disorders. EBNA1 is the viral-encoded DNA binding protein required for episomal maintenance during latent infection and is consistently expressed in all EBV tumors. The interaction of EBNA1 with different genetic elements confers different viral functions, such as replication initiation at DS and chromosome tethering at FR. Here, we used cryo-EM to determine the structure of the EBNA1 DNA-binding domain (DBD) bound to two tandem sites at the DS and at the FR. We also show that the NTD of EBNA1 can interact with the AT-rich DNA sequence between tandem EBNA1 DBD binding sites in the FR. These results provide new information on the mechanism of EBNA1 DNA binding at DS and FR and suggest a higher-order oligomeric structure of EBNA1 bound to FR. Our findings have implications for targeting EBNA1 in EBV-associated disease.
History
DepositionJul 7, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 17, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Epstein-Barr nuclear antigen 1
B: Epstein-Barr nuclear antigen 1
C: Epstein-Barr nuclear antigen 1
D: Epstein-Barr nuclear antigen 1
E: 2XFR DNA (56-MER)
F: 2XFR DNA (56-MER)


Theoretical massNumber of molelcules
Total (without water)104,7636
Polymers104,7636
Non-polymers00
Water724
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, 0.125% glutaraldehyde was used in ultracentrifuge to facilitate the assembly, Ultracentrifuge: 40000rpm, 16hrs
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Epstein-Barr nuclear antigen 1 / EBNA-1 / EBV nuclear antigen 1


Mass: 17564.369 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human herpesvirus 4 strain B95-8 (Epstein-Barr virus (strain B95-8))
Strain: B95-8 / Gene: EBNA1, BKRF1 / Plasmid: pET-mod / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03211
#2: DNA chain 2XFR DNA (56-MER)


Mass: 17241.064 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human herpesvirus 4 strain B95-8 (Epstein-Barr virus (strain B95-8))
Production host: chemical production metagenome (others)
#3: DNA chain 2XFR DNA (56-MER)


Mass: 17264.139 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human herpesvirus 4 strain B95-8 (Epstein-Barr virus (strain B95-8))
Production host: chemical production metagenome (others)
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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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: EBNA1 DBD+2XFR complex / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Human herpesvirus 4 strain B95-8 (Epstein-Barr virus (strain B95-8))
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2100 mMsodium chlorideNaCl1
32 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 22 % / Chamber temperature: 293 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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 105000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 10000 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 1.25 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5300

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Processing

SoftwareName: PHENIX / Version: 1.20_4444: / Classification: refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1242375 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0045015
ELECTRON MICROSCOPYf_angle_d0.6816807
ELECTRON MICROSCOPYf_dihedral_angle_d25.8632975
ELECTRON MICROSCOPYf_chiral_restr0.3361177
ELECTRON MICROSCOPYf_plane_restr0.007999

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