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- EMDB-41736: Cryo-EM structure of bovine concentrative nucleoside transporter ... -

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Basic information

Entry
Database: EMDB / ID: EMD-41736
TitleCryo-EM structure of bovine concentrative nucleoside transporter 3 in complex with Molnupiravir, condition 1, INT1-INT1-INT1 conformation
Map datafull map
Sample
  • Complex: bCNT3 trimer
    • Protein or peptide: Sodium/nucleoside cotransporter
  • Ligand: N-hydroxy-5'-O-(2-methylpropanoyl)cytidine
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
Keywordsmembrane protein / transporter / nucleoside / TRANSPORT PROTEIN
Function / homology
Function and homology information


Ribavirin ADME / pyrimidine- and adenosine-specific:sodium symporter activity / purine-specific nucleoside:sodium symporter activity / Transport of nucleosides and free purine and pyrimidine bases across the plasma membrane / pyrimidine nucleoside transport / uridine transmembrane transporter activity / purine nucleoside transmembrane transport / Azathioprine ADME / brush border membrane / plasma membrane
Similarity search - Function
Concentrative nucleoside transporter N-terminal domain / Concentrative nucleoside transporter / Concentrative nucleoside transporter C-terminal domain / Concentrative nucleoside transporter, metazoan/bacterial / Na+ dependent nucleoside transporter N-terminus / Na+ dependent nucleoside transporter C-terminus / Nucleoside transporter/FeoB GTPase, Gate domain / Nucleoside recognition
Similarity search - Domain/homology
Sodium/nucleoside cotransporter
Similarity search - Component
Biological speciesBos taurus (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsWright NJ / Lee S-Y
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R21AI166134 United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Antiviral drug recognition and elevator-type transport motions of CNT3.
Authors: Nicholas J Wright / Feng Zhang / Yang Suo / Lingyang Kong / Ying Yin / Justin G Fedor / Kedar Sharma / Mario J Borgnia / Wonpil Im / Seok-Yong Lee /
Abstract: Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human ...Nucleoside analogs have broad clinical utility as antiviral drugs. Key to their systemic distribution and cellular entry are human nucleoside transporters. Here, we establish that the human concentrative nucleoside transporter 3 (CNT3) interacts with antiviral drugs used in the treatment of coronavirus infections. We report high-resolution single-particle cryo-electron microscopy structures of bovine CNT3 complexed with antiviral nucleosides N-hydroxycytidine, PSI-6206, GS-441524 and ribavirin, all in inward-facing states. Notably, we found that the orally bioavailable antiviral molnupiravir arrests CNT3 in four distinct conformations, allowing us to capture cryo-electron microscopy structures of drug-loaded outward-facing and drug-loaded intermediate states. Our studies uncover the conformational trajectory of CNT3 during membrane transport of a nucleoside analog antiviral drug, yield new insights into the role of interactions between the transport and the scaffold domains in elevator-like domain movements during drug translocation, and provide insights into the design of nucleoside analog antiviral prodrugs with improved oral bioavailability.
History
DepositionAug 26, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41736.png

Downloads & links

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Map

FileDownload / File: emd_41736.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfull map
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-2.3270884 - 3.3758526
Average (Standard dev.)0.0017266284 (±0.0833024)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map 1

Fileemd_41736_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_41736_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 : bCNT3 trimer

EntireName: bCNT3 trimer
Components
  • Complex: bCNT3 trimer
    • Protein or peptide: Sodium/nucleoside cotransporter
  • Ligand: N-hydroxy-5'-O-(2-methylpropanoyl)cytidine
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

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Supramolecule #1: bCNT3 trimer

SupramoleculeName: bCNT3 trimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 232 KDa

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Macromolecule #1: Sodium/nucleoside cotransporter

MacromoleculeName: Sodium/nucleoside cotransporter / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 79.376773 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDYKDDDDKL EATMAMSSKI SVELQRVAAL PAQGCSNTGF QNDEDGFENQ NPSGNDHSLR NRVVQNREHE NGKQVEEHIT IGQDSLRKD EEEEDDQETH RKGCLERMCG RMSDFCREHK TTLRYIIWGI LIAGYLALVI AACVMNFHRA LPLFVITVVA I FFVVWDHL ...String:
MDYKDDDDKL EATMAMSSKI SVELQRVAAL PAQGCSNTGF QNDEDGFENQ NPSGNDHSLR NRVVQNREHE NGKQVEEHIT IGQDSLRKD EEEEDDQETH RKGCLERMCG RMSDFCREHK TTLRYIIWGI LIAGYLALVI AACVMNFHRA LPLFVITVVA I FFVVWDHL MAKYESQIAR FLSPGQRLLD SHWFWLKWVI WGCLILGVIL WLVFDTAKLG QQQLVSFGGL IIYTSLTFLF SK HPTKVYW RPVFWGIGLQ FLLGLLILRT EPGFMAFDWL GKQVQTFLGY SDAGASFVFG EKYTDHFFAF KVLPIVIFFS TVM SMLYYL GLMQWIIRKV GWVMLVTMGT SPVESVVASG NIFIGQTESP LLVRPYLPYV TKSELHAIMT AGFSTIAGSV LGAY ISFGV SSSHLLTASV MSAPAALAIS KLFWPETETP KINLKNAMKM ESGDSRNLLE AATQGASSSI SLVANIAVNL IAFLA LLSF MNSALSWLGN MFDYPQLSFE VICSYVFMPF AFMMGVDWQD SFMVAKLIGY KTFFNEFVAY QQLSKLISLR QVGGPK FVD GVQQYMSMRS EAISTYALCG FANFGSLGIV IGGLTSMAPS RKRDITAGAM RALIAGTIAC FLTACIAGML TNTPVDI NC HHILENAFNS GLVRNTTNVV SCCQGLLSSA VVKGPGEVIP TGNHSLYSLK NCCNLLNTPT LNCSWIPNVL SNS

UniProtKB: Sodium/nucleoside cotransporter

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Macromolecule #2: N-hydroxy-5'-O-(2-methylpropanoyl)cytidine

MacromoleculeName: N-hydroxy-5'-O-(2-methylpropanoyl)cytidine / type: ligand / ID: 2 / Number of copies: 3 / Formula: XMO
Molecular weightTheoretical: 329.306 Da

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Macromolecule #3: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

MacromoleculeName: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / type: ligand / ID: 3 / Number of copies: 3 / Formula: LBN
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-LBN:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / phospholipid*YM / POPC

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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

Concentration8 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
150.0 mMNaClSodium chloride
20.0 mMTris
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 4715 / Average exposure time: 2.4 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2592142
Startup modelType of model: OTHER
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.3)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 3.3)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 3.3)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3) / Number images used: 127798

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Atomic model buiding 1

Initial modelPDB ID:

3tij


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8tz7:
Cryo-EM structure of bovine concentrative nucleoside transporter 3 in complex with Molnupiravir, condition 1, INT1-INT1-INT1 conformation

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