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- PDB-6s3a: Coxsackie B3 2C protein in complex with S-Fluoxetine -

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Basic information

Entry
Database: PDB / ID: 6s3a
TitleCoxsackie B3 2C protein in complex with S-Fluoxetine
Components2C protein
KeywordsVIRAL PROTEIN / 2C / enterovirus / helicase / ATPase / complex / antiviral / Porzac / fluoxetine / repurposing / coxsackievirus
Function / homology
Function and homology information


: / symbiont-mediated perturbation of host gene expression / RNA-protein covalent cross-linking / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / ribonucleoside triphosphate phosphatase activity / symbiont genome entry into host cell via pore formation in plasma membrane ...: / symbiont-mediated perturbation of host gene expression / RNA-protein covalent cross-linking / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MDA-5 activity / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of MAVS activity / picornain 2A / symbiont-mediated suppression of host mRNA export from nucleus / ribonucleoside triphosphate phosphatase activity / symbiont genome entry into host cell via pore formation in plasma membrane / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / endocytosis involved in viral entry into host cell / : / nucleoside-triphosphate phosphatase / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity / DNA replication / induction by virus of host autophagy / RNA-directed RNA polymerase / symbiont-mediated suppression of host gene expression / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / host cell nucleus / virion attachment to host cell / structural molecule activity / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / membrane / metal ion binding
Similarity search - Function
Picornavirus coat protein VP4 superfamily / Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) ...Picornavirus coat protein VP4 superfamily / Poliovirus 3A protein-like / Poliovirus 3A protein like / Picornavirus 2B protein / Poliovirus core protein 3a, soluble domain / Picornavirus 2B protein / Peptidase C3, picornavirus core protein 2A / Picornavirus core protein 2A / Picornavirus coat protein VP4 / Picornavirus coat protein (VP4) / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-SFX / Genome polyprotein / Genome polyprotein
Similarity search - Component
Biological speciesCoxsackievirus B3
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.52 Å
AuthorsEl Kazzi, P. / Papageorgiou, N. / Ferron, F.P. / Bauer, L. / van Kuppeveld, F. / Coutard, B.
Funding support1items
OrganizationGrant numberCountry
European Commission642434
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
DepositionJun 24, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 1, 2022Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: 2C protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,1744
Polymers23,7631
Non-polymers4103
Water3,801211
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area110 Å2
ΔGint-7 kcal/mol
Surface area11210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)48.398, 53.183, 79.152
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein 2C protein


Mass: 23763.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Coxsackievirus B3 / Production host: Escherichia coli (E. coli) / Strain (production host): T7 Express Iq (Biolabs) / References: UniProt: Q9E7C2, UniProt: P03313*PLUS
#2: Chemical ChemComp-SFX / (3S)-N-methyl-3-phenyl-3-[4-(trifluoromethyl)phenoxy]propan-1-amine / Fluoxetine / Fluoxetine


Mass: 309.326 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H18F3NO / Feature type: SUBJECT OF INVESTIGATION / Comment: antidepressant, inhibitor*YM
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 211 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.14 Å3/Da / Density % sol: 42.61 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.5 / Details: Mes 0.1M pH 6,5, PEG 5000 27, 2M ammonium sulfate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: Y
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: BM30A / Wavelength: 0.976251 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Sep 10, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.976251 Å / Relative weight: 1
ReflectionResolution: 1.52→44.14 Å / Num. obs: 33298 / % possible obs: 98.18 % / Redundancy: 5.5 % / Rmerge(I) obs: 0.07759 / Rpim(I) all: 0.0326 / Rrim(I) all: 0.07759 / Net I/σ(I): 13.69
Reflection shellResolution: 1.52→1.57 Å / Redundancy: 5.4 % / Rmerge(I) obs: 0.8 / Mean I/σ(I) obs: 1.89 / Num. unique obs: 3154 / Rpim(I) all: 0.39 / Rrim(I) all: 0.9 / % possible all: 94.87
Serial crystallography sample deliveryMethod: fixed target

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Processing

Software
NameVersionClassification
PHENIX1.13refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.52→44.14 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflection
Rfree0.1995 --
obs0.1853 31556 98.18 %
Refinement stepCycle: LAST / Resolution: 1.52→44.14 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1607 0 24 211 1842

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