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- PDB-7opl: CryoEM structure of DNA Polymerase alpha - primase bound to SARS ... -

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Basic information

Entry
Database: PDB / ID: 7opl
TitleCryoEM structure of DNA Polymerase alpha - primase bound to SARS CoV nsp1
Components
  • (DNA polymerase alpha ...DNA polymerase) x 2
  • DNA primase large subunitPrimase
  • DNA primase small subunitPrimase
  • Non-structural protein 1
KeywordsDNA BINDING PROTEIN / DNA polymerase / Primase / viral protein
Function / homology
Function and homology information


DNA primase AEP / positive regulation of DNA primase activity / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / Polymerase switching / alpha DNA polymerase:primase complex / regulation of type I interferon production ...DNA primase AEP / positive regulation of DNA primase activity / ribonucleotide binding / DNA replication initiation / DNA/RNA hybrid binding / Telomere C-strand synthesis initiation / Inhibition of replication initiation of damaged DNA by RB1/E2F1 / Polymerase switching / alpha DNA polymerase:primase complex / regulation of type I interferon production / Processive synthesis on the lagging strand / Assembly of the SARS-CoV-1 Replication-Transcription Complex (RTC) / Maturation of replicase proteins / Removal of the Flap Intermediate / Transcription of SARS-CoV-1 sgRNAs / DNA primase activity / Polymerase switching on the C-strand of the telomere / lagging strand elongation / mitotic DNA replication initiation / DNA replication, synthesis of primer / Translation of Replicase and Assembly of the Replication Transcription Complex / Replication of the SARS-CoV-1 genome / K48-linked deubiquitinase activity / DNA strand elongation involved in DNA replication / host cell endoplasmic reticulum / K63-linked deubiquitinase activity / leading strand elongation / DNA synthesis involved in DNA repair / G1/S-Specific Transcription / DNA replication origin binding / SARS-CoV-1 modulates host translation machinery / DNA replication initiation / Activation of the pre-replicative complex / viral genome replication / Defective pyroptosis / methyltransferase activity / nuclear matrix / double-strand break repair via nonhomologous end joining / protein import into nucleus / SARS-CoV-1 activates/modulates innate immune responses / single-stranded DNA binding / nuclear envelope / 4 iron, 4 sulfur cluster binding / methylation / double membrane vesicle viral factory outer membrane / SARS coronavirus main proteinase / host cell endosome / symbiont-mediated degradation of host mRNA / mRNA guanylyltransferase / symbiont-mediated suppression of host ISG15-protein conjugation / G-quadruplex RNA binding / omega peptidase activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of IRF3 activity / host cell Golgi apparatus / symbiont-mediated perturbation of host ubiquitin-like protein modification / endonuclease activity / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / Hydrolases; Acting on peptide bonds (peptidases); Cysteine endopeptidases / DNA replication / DNA-directed DNA polymerase / single-stranded RNA binding / host cell perinuclear region of cytoplasm / DNA-directed DNA polymerase activity / viral protein processing / lyase activity / induction by virus of host autophagy / symbiont-mediated suppression of host gene expression / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / nucleotide binding / DNA repair / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / chromatin binding / chromatin / nucleolus / protein kinase binding / magnesium ion binding / proteolysis / DNA binding / zinc ion binding / nucleoplasm / membrane / identical protein binding / metal ion binding / nucleus / cytosol
Similarity search - Function
DNA polymerase alpha, subunit B, N-terminal domain superfamily / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain ...DNA polymerase alpha, subunit B, N-terminal domain superfamily / DNA polymerase alpha subunit B N-terminal / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / DNA primase, small subunit, eukaryotic/archaeal / DNA primase, large subunit, eukaryotic / DNA primase, small subunit / DNA primase small subunit / DNA primase large subunit, eukaryotic/archaeal / DNA polymerase alpha catalytic subunit, N-terminal domain / DNA polymerase alpha, zinc finger domain superfamily / Eukaryotic and archaeal DNA primase, large subunit / DNA Polymerase alpha zinc finger / DNA polymerase alpha subunit p180 N terminal / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha catalytic subunit, catalytic domain / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / Non-structural protein 3, SUD-N macrodomain, SARS-CoV / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Non-structural protein NSP3, SUD-N (Mac2) domain, betacoronavirus / Sarbecovirus Nsp3c-N domain profile. / Non-structural protein NSP3, N-terminal, betacoronavirus / Polyprotein cleavage domain PL2pro superfamily, betacoronavirus / Non-structural protein NSP3, SUD-N (Mac2) domain superfamily, betacoronavirus / Betacoronavirus SUD-C domain / Betacoronavirus replicase NSP3, N-terminal / NSP1 globular domain superfamily, betacoronavirus / Non-structural protein 2, SARS-CoV-like / Coronavirus 3Ecto domain profile. / : / Betacoronavirus Nsp3e group 2-specific marker (G2M) domain profile. / NSP1, C-terminal domain, betacoronavirus / Betacoronavirus Nsp3c-M domain profile. / NSP1, globular domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain, betacoronavirus / Non-structural protein NSP3, SUD-M domain superfamily, betacoronavirus / Betacoronavirus replicase NSP1 / Betacoronavirus single-stranded poly(A) binding domain / Betacoronavirus (BetaCoV) Nsp1 C-terminal domain profile. / Betacoronavirus Nsp3c-C domain profile. / Betacoronavirus Nsp3e nucleic acid-binding (NAB) domain profile. / DPUP/SUD, C-terminal, betacoronavirus / Non-structural protein NSP3, nucleic acid-binding domain superfamily, betacoronavirus / Non-structural protein 6, betacoronavirus / Betacoronavirus nucleic acid-binding (NAB) / Non-structural protein NSP3, nucleic acid-binding domain, betacoronavirus / Non-structural protein NSP3A domain-like superfamily / Papain-like protease, N-terminal domain superfamily, coronavirus / Papain-like viral protease, palm and finger domains, coronavirus / : / Coronavirus (CoV) Nsp2 middle domain profile. / Coronavirus (CoV) Nsp2 N-terminal domain profile. / Coronavirus (CoV) Nsp2 C-terminal domain profile. / NSP1, globular domain, alpha/betacoronavirus / : / Coronavirus (CoV) Nsp3 Y domain profile. / Coronavirus (CoV) Nsp1 globular domain profile. / Coronavirus replicase NSP2, N-terminal / Nonstructural protein 2, N-terminal domain, coronavirus / Coronavirus replicase NSP2, C-terminal / Non-structural protein 2, C-terminal domain, coronavirus / Coronavirus Nsp3a Ubl domain profile. / Coronavirus Nsp3d Ubl domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp7 cofactor domain profile. / Coronavirus RNA-dependent RNA polymerase (RdRp) Nsp8 cofactor domain profile. / Coronavirus Nsp9 single-stranded RNA (ssRNA)-binding domain profile. / Coronavirus (CoV) ExoN/MTase coactivator domain profile. / NSP3, first ubiquitin-like (Ubl) domain, coronavirus / NSP3, second ubiquitin-like (Ubl) domain, coronavirus / Coronavirus Nsp4 C-terminal (Nsp4C) domain profile. / Papain-like protease, thumb domain superfamily, coronavirus / Coronavirus replicase NSP7 / Peptidase family C16 domain profile. / Non-structural protein NSP7, coronavirus / Peptidase C30, coronavirus / Peptidase C16, coronavirus / Non-structural protein NSP9, coronavirus / Non-structural protein NSP8, coronavirus / RNA synthesis protein NSP10, coronavirus / Non-structural protein NSP4, C-terminal, coronavirus / RNA synthesis protein NSP10 superfamily, coronavirus / Non-structural protein NSP9 superfamily, coronavirus / Non-structural protein NSP7 superfamily, coronavirus / Non-structural protein NSP8 superfamily, coronavirus / Non-structural protein NSP4, C-terminal superfamily, coronavirus / Peptidase C30, domain 3, coronavirus / Non-structural protein 6, coronavirus / Coronavirus replicase NSP3, C-terminal / Non-structural protein NSP4, N-terminal, coronavirus / Coronavirus endopeptidase C30
Similarity search - Domain/homology
IRON/SULFUR CLUSTER / DNA polymerase alpha catalytic subunit / Replicase polyprotein 1a / DNA primase small subunit / DNA primase large subunit / DNA polymerase alpha subunit B
Similarity search - Component
Biological speciesHomo sapiens (human)
Severe acute respiratory syndrome coronavirus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.12 Å
AuthorsKilkenny, M.L. / Pellegrini, L.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust104641/Z/14/Z United Kingdom
CitationJournal: Protein Sci / Year: 2022
Title: Structural basis for the interaction of SARS-CoV-2 virulence factor nsp1 with DNA polymerase α-primase.
Authors: Mairi L Kilkenny / Charlotte E Veale / Amir Guppy / Steven W Hardwick / Dimitri Y Chirgadze / Neil J Rzechorzek / Joseph D Maman / Luca Pellegrini /
Abstract: The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense ...The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-the causative agent of coronavirus disease 2019 (COVID-19)-are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS-CoV-2. The DNA polymerase α (Pol α)-primase complex or primosome-responsible for initiating DNA synthesis during genomic duplication-was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS-CoV-2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo-electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS-CoV-2 interferes with Pol α's putative role in the immune response during the viral infection.
History
DepositionJun 1, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 10, 2021Provider: repository / Type: Initial release
Revision 1.1Nov 24, 2021Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc / Item: _citation.title / _em_admin.last_update
Revision 1.2Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: DNA polymerase alpha catalytic subunit
B: DNA polymerase alpha subunit B
C: DNA primase small subunit
D: DNA primase large subunit
E: Non-structural protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)308,5439
Polymers307,9965
Non-polymers5484
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: The Pol alpha-primase complex forms a constitutive hetero-tetramer. The interaction with nsp1 was demostrated by pulldown experiments with MBP-tagged nsp1 protein.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14830 Å2
ΔGint-84 kcal/mol
Surface area109090 Å2
MethodPISA

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Components

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DNA polymerase alpha ... , 2 types, 2 molecules AB

#1: Protein DNA polymerase alpha catalytic subunit / DNA polymerase alpha catalytic subunit p180


Mass: 133702.562 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA1, POLA / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P09884, DNA-directed DNA polymerase
#2: Protein DNA polymerase alpha subunit B / DNA polymerase / DNA polymerase alpha 70 kDa subunit


Mass: 49855.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: POLA2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q14181

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Protein , 3 types, 3 molecules CDE

#3: Protein DNA primase small subunit / Primase / DNA primase 49 kDa subunit / p49


Mass: 52590.801 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P49642, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#4: Protein DNA primase large subunit / Primase / DNA primase 58 kDa subunit / p58


Mass: 58890.918 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PRIM2, PRIM2A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P49643
#5: Protein Non-structural protein 1 / nsp1 / Leader protein


Mass: 12955.852 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Severe acute respiratory syndrome coronavirus
Gene: 1a / Production host: Escherichia coli (E. coli) / References: UniProt: P0C6U8

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Non-polymers , 2 types, 4 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-SF4 / IRON/SULFUR CLUSTER / Iron–sulfur cluster


Mass: 351.640 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe4S4

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Details

Has ligand of interestN

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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: Complex of DNA polymerase alpha - primase bound to SARS COV-2 nsp1
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.31 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.2
Buffer component
IDConc.NameBuffer-ID
125 mMHepes1
2150 mMKCl1
31 1mMDTT1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The nsp1 protein was added in 10-fold stoichiometric excess
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: -0.7 nm / Nominal defocus min: -2.5 nm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.31 sec. / Electron dose: 46.91 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 2919

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPU2.7image acquisition
4CTFFIND4.1CTF correction
7PHENIX1.19.1-4122model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIX1.19.1-4122model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 709068
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.12 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 233476 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
15EXR

5exr

A1
25EXR

5exr

B1
35EXR

5exr

C1
45EXR

5exr

D1
57K3N

7k3n

E1
62HSX

2hsx

E1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320189
ELECTRON MICROSCOPYf_angle_d0.72827293
ELECTRON MICROSCOPYf_dihedral_angle_d5.2362659
ELECTRON MICROSCOPYf_chiral_restr0.0473003
ELECTRON MICROSCOPYf_plane_restr0.0073506

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