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- EMDB-12798: Hexameric coxsackievirus B3 2C protein in complex with S-fluoxetine -

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Basic information

Entry
Database: EMDB / ID: EMD-12798
TitleHexameric coxsackievirus B3 2C protein in complex with S-fluoxetine
Map dataHexameric coxsackievirus B3 2C protein in complex with S-fluoxetine
Sample
  • Complex: Hexameric coxsackievirus B3 2C protein in complex with S-fluoxetine
    • Protein or peptide: Coxsackievirus B3 2C protein
Biological speciesCoxsackievirus B3 (strain Nancy)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.0 Å
AuthorsHurdiss DL / Forster F
Funding support Netherlands, 5 items
OrganizationGrant numberCountry
H2020 Marie Curie Actions of the European Commission842333 Netherlands
Netherlands Organisation for Scientific Research (NWO)ECHO-711.017.002 Netherlands
H2020 Marie Curie Actions of the European Commission642434 Netherlands
Netherlands Organisation for Scientific Research (NWO)NWO-VICI-91812628 Netherlands
European Research Council (ERC)724425 Netherlands
CitationJournal: Sci Adv / Year: 2022
Title: Fluoxetine targets an allosteric site in the enterovirus 2C AAA+ ATPase and stabilizes a ring-shaped hexameric complex.
Authors: Daniel L Hurdiss / Priscila El Kazzi / Lisa Bauer / Nicolas Papageorgiou / François P Ferron / Tim Donselaar / Arno L W van Vliet / Tatiana M Shamorkina / Joost Snijder / Bruno Canard / ...Authors: Daniel L Hurdiss / Priscila El Kazzi / Lisa Bauer / Nicolas Papageorgiou / François P Ferron / Tim Donselaar / Arno L W van Vliet / Tatiana M Shamorkina / Joost Snijder / Bruno Canard / Etienne Decroly / Andrea Brancale / Tzviya Zeev-Ben-Mordehai / Friedrich Förster / Frank J M van Kuppeveld / Bruno Coutard /
Abstract: Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing ...Enteroviruses are globally prevalent human pathogens responsible for many diseases. The nonstructural protein 2C is a AAA+ helicase and plays a key role in enterovirus replication. Drug repurposing screens identified 2C-targeting compounds such as fluoxetine and dibucaine, but how they inhibit 2C is unknown. Here, we present a crystal structure of the soluble and monomeric fragment of coxsackievirus B3 2C protein in complex with ()-fluoxetine (SFX), revealing an allosteric binding site. To study the functional consequences of SFX binding, we engineered an adenosine triphosphatase (ATPase)–competent, hexameric 2C protein. Using this system, we show that SFX, dibucaine, HBB [2-(α-hydroxybenzyl)-benzimidazole], and guanidine hydrochloride inhibit 2C ATPase activity. Moreover, cryo–electron microscopy analysis demonstrated that SFX and dibucaine lock 2C in a defined hexameric state, rationalizing their mode of inhibition. Collectively, these results provide important insights into 2C inhibition and a robust engineering strategy for structural, functional, and drug-screening analysis of 2C proteins.
History
DepositionApr 22, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateJun 1, 2022-
Current statusJun 1, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0661
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0661
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12798.png

Downloads & links

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Map

FileDownload / File: emd_12798.map.gz / Format: CCP4 / Size: 2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHexameric coxsackievirus B3 2C protein in complex with S-fluoxetine
Voxel sizeX=Y=Z: 2.76 Å
Density
Contour LevelBy AUTHOR: 0.0661 / Movie #1: 0.0661
Minimum - Maximum-0.021918446 - 0.12473615
Average (Standard dev.)0.0022112278 (±0.011141417)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 220.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.762.762.76
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z220.800220.800220.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0220.1250.002

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Supplemental data

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Half map: Half map 1

Fileemd_12798_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_12798_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hexameric coxsackievirus B3 2C protein in complex with S-fluoxetine

EntireName: Hexameric coxsackievirus B3 2C protein in complex with S-fluoxetine
Components
  • Complex: Hexameric coxsackievirus B3 2C protein in complex with S-fluoxetine
    • Protein or peptide: Coxsackievirus B3 2C protein

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Supramolecule #1: Hexameric coxsackievirus B3 2C protein in complex with S-fluoxetine

SupramoleculeName: Hexameric coxsackievirus B3 2C protein in complex with S-fluoxetine
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Coxsackievirus B3 (strain Nancy)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta / Recombinant plasmid: pET28b
Molecular weightExperimental: 170 KDa

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Macromolecule #1: Coxsackievirus B3 2C protein

MacromoleculeName: Coxsackievirus B3 2C protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: nucleoside-triphosphate phosphatase
Source (natural)Organism: Coxsackievirus B3 (strain Nancy)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPGGGGSGGG GSGELKAIAQ ELKAIAKELK AIAWELKAIA QGAGGSGSYF QSNASKCRIE PVCLLLHGSP GAGKSVATNL IGRSLAEKLN SSVYSLPPDP DHFDGYKQQA VVIMDDLCQN PDGKDVSLFC QMVSSVDFVP PMAALEEKGI LFTSPFVLAS TNAGSINAPT ...String:
GPGGGGSGGG GSGELKAIAQ ELKAIAKELK AIAWELKAIA QGAGGSGSYF QSNASKCRIE PVCLLLHGSP GAGKSVATNL IGRSLAEKLN SSVYSLPPDP DHFDGYKQQA VVIMDDLCQN PDGKDVSLFC QMVSSVDFVP PMAALEEKGI LFTSPFVLAS TNAGSINAPT VSDSRALARR FHFDMNIEVI SMYSQNGKIN MPMSVKTCDD ECCPVNFKKC CPLVCGKAIQ FIDRRTQVRY SLDMLVTEMF REYNHRHSVG TTLEALFQ

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
25.0 millimolarC4H11NO3Tris
300.0 millimolarNaClSodium chlorideSodium chloride
1.0 millimolarMgCl2Magnesium chloride
0.5 PercentC2H6OSDimethyl sulfoxide
100.0 micromolarC17H18F3NO(S)-Fluoxetine
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Details: 20 mA using a PELCO easyGLow
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsTo account for the preferred orientation exhibited by the sample an alpha tilt of +30 degrees was used for this data collection.
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 6119 / Average exposure time: 4.0 sec. / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 180069
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.1) / Number images used: 6856
FSC plot (resolution estimation)

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