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- PDB-8v1t: Herpes simplex virus 1 polymerase holoenzyme bound to DNA and acy... -

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Basic information

Entry
Database: PDB / ID: 8v1t
TitleHerpes simplex virus 1 polymerase holoenzyme bound to DNA and acyclovir triphosphate in closed conformation
Components
  • DNA polymerase
  • DNA polymerase processivity factor
  • PRIMER DNA (32-MER)
  • TEMPLATE DNA (50-MER)
KeywordsTRANSFERASE/DNA / herpes simplex virus / replication / DNA polymerase / ACYCLOVIR / antiherpesvirus drug / TRANSFERASE-DNA complex
Function / homology
Function and homology information


viral DNA genome replication / DNA-templated DNA replication / RNA-DNA hybrid ribonuclease activity / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / host cell nucleus / DNA binding
Similarity search - Function
DNA polymerase processivity factor (UL42) / DNA polymerase processivity factor (UL42) / DNA polymerase catalytic subunit Pol, C-terminal / DNA polymerase catalytic subunit Pol / : / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B ...DNA polymerase processivity factor (UL42) / DNA polymerase processivity factor (UL42) / DNA polymerase catalytic subunit Pol, C-terminal / DNA polymerase catalytic subunit Pol / : / DNA polymerase family B, thumb domain / DNA-directed DNA polymerase, family B, multifunctional 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 polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
ACYCLOVIR TRIPHOSPHATE / DNA / DNA (> 10) / DNA polymerase / DNA polymerase processivity factor
Similarity search - Component
Biological speciesHuman alphaherpesvirus 1 strain KOS
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsPan, J. / Abraham, J. / Coen, D.M. / Shankar, S. / Yang, P. / Hogle, J.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R21 AI141940 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01 AI019838 United States
CitationJournal: Cell / Year: 2024
Title: Viral DNA polymerase structures reveal mechanisms of antiviral drug resistance.
Authors: Sundaresh Shankar / Junhua Pan / Pan Yang / Yuemin Bian / Gábor Oroszlán / Zishuo Yu / Purba Mukherjee / David J Filman / James M Hogle / Mrinal Shekhar / Donald M Coen / Jonathan Abraham /
Abstract: DNA polymerases are important drug targets, and many structural studies have captured them in distinct conformations. However, a detailed understanding of the impact of polymerase conformational ...DNA polymerases are important drug targets, and many structural studies have captured them in distinct conformations. However, a detailed understanding of the impact of polymerase conformational dynamics on drug resistance is lacking. We determined cryoelectron microscopy (cryo-EM) structures of DNA-bound herpes simplex virus polymerase holoenzyme in multiple conformations and interacting with antivirals in clinical use. These structures reveal how the catalytic subunit Pol and the processivity factor UL42 bind DNA to promote processive DNA synthesis. Unexpectedly, in the absence of an incoming nucleotide, we observed Pol in multiple conformations with the closed state sampled by the fingers domain. Drug-bound structures reveal how antivirals may selectively bind enzymes that more readily adopt the closed conformation. Molecular dynamics simulations and the cryo-EM structure of a drug-resistant mutant indicate that some resistance mutations modulate conformational dynamics rather than directly impacting drug binding, thus clarifying mechanisms that drive drug selectivity.
History
DepositionNov 21, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 11, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Sep 18, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.3Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: citation / em_admin / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _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: DNA polymerase
B: DNA polymerase processivity factor
P: PRIMER DNA (32-MER)
T: TEMPLATE DNA (50-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)195,5899
Polymers195,0274
Non-polymers5625
Water724
1


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

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Components

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Protein , 2 types, 2 molecules AB

#1: Protein DNA polymerase


Mass: 133614.344 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Herpes simplex virus type 1 (KOS strain) DNA polymerase catalytic subunit UL30 with its N-terminal 42 residues deleted and replaced by an N-terminal poly-histidine tag in the expression construct
Source: (gene. exp.) Human alphaherpesvirus 1 strain KOS / Gene: UL30, HHV1gp046 / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: H9E937, DNA-directed DNA polymerase
#2: Protein DNA polymerase processivity factor / Polymerase accessory protein


Mass: 36346.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Herpes simplex virus 1 (KOS strain) DNA polymerase processivity factor UL42 residues 1-340 tagged with a prescission protease cleavage site followed by a maltose binding proten (MBP) tag in ...Details: Herpes simplex virus 1 (KOS strain) DNA polymerase processivity factor UL42 residues 1-340 tagged with a prescission protease cleavage site followed by a maltose binding proten (MBP) tag in the expression construct
Source: (gene. exp.) Human alphaherpesvirus 1 strain KOS / Gene: UL42, HHV1gp061 / Plasmid: PMAL-C2 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): PLYSS / References: UniProt: H9E949

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DNA chain , 2 types, 2 molecules PT

#3: DNA chain PRIMER DNA (32-MER)


Mass: 9866.353 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: 3'-deoxyl primer DNA strand, no mismatch with template strand
Source: (synth.) synthetic construct (others)
#4: DNA chain TEMPLATE DNA (50-MER)


Mass: 15199.773 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 3 types, 9 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-AVP / ACYCLOVIR TRIPHOSPHATE


Mass: 465.144 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H14N5O12P3 / Feature type: SUBJECT OF INVESTIGATION
#7: 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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Details

Has ligand of interestY
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: HERPES SIMPLEX VIRUS TYPE 1 POLYMERASE HOLOENZYME UL30:UL42 IN COMPLEX WITH dsDNA AND ACYCLOVIR TRIPHOSPHATE
Type: COMPLEX
Details: EACH COMPLEX CONSISTS OF ONE HSV-1 UL30 (expressed in insect cells), ONE HSV-1 UL42 (expressed in E.coli), ONE TEMPLATE DNA (synthetic), ONE PRIMER DNA (synthetic), AND ONE ACYCLOVIR ...Details: EACH COMPLEX CONSISTS OF ONE HSV-1 UL30 (expressed in insect cells), ONE HSV-1 UL42 (expressed in E.coli), ONE TEMPLATE DNA (synthetic), ONE PRIMER DNA (synthetic), AND ONE ACYCLOVIR TRIPHOSPHATE MOLECULE (synthetic).
Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.197 MDa / Experimental value: NO
Source (natural)Organism: Human alphaherpesvirus 1 strain KOS / Strain: KOS
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: SF9
Buffer solutionpH: 7.5
Details: 25 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM tris(2-carboxyethyl)phosphine (TCEP)
SpecimenConc.: 1.38 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: GRIDS WERE GLOW DISCHARGED IN A PELCO EASIGLOW AT 15 MA FOR 30 SECONDS UNDER 0.39 MBAR.
Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
Details: 3 MICROLITERS OF SAMPLE WERE BLOTTED FOR 3 SECONDS WITH FILTER PAPER SATURATED UNDER 100% HUMIDITY PRIOR TO PLUNGING.

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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: 105000 X / Calibrated magnification: 60606 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 77 K / Temperature (min): 70 K
Image recordingElectron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3268
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1crYOLO1.7.5particle selection
2SerialEM3.8image acquisition
4CTFFIND4.1.10CTF correction
7UCSF Chimera1.13.1model fitting
8O15.1.0model fitting
10PHENIX1.20.1-4487model refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1214260
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 187300 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 33.64 / Protocol: OTHER / Space: REAL / Target criteria: CORRELATION COEFFICIENT
Details: RIGID BODY, MINIMIZATION_GLOBAL, LOCAL_GRID_SEARCH, ADP REFINEMENT
RefinementHighest resolution: 2.8 Å

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