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- PDB-8tz7: Cryo-EM structure of bovine concentrative nucleoside transporter ... -

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Basic information

Entry
Database: PDB / ID: 8tz7
TitleCryo-EM structure of bovine concentrative nucleoside transporter 3 in complex with Molnupiravir, condition 1, INT1-INT1-INT1 conformation
ComponentsSodium/nucleoside cotransporter
KeywordsTRANSPORT PROTEIN / membrane protein / transporter / nucleoside
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
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / : / Sodium/nucleoside cotransporter
Similarity search - Component
Biological speciesBos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsWright, N.J. / Lee, S.-Y.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium/nucleoside cotransporter
B: Sodium/nucleoside cotransporter
C: Sodium/nucleoside cotransporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)241,3989
Polymers238,1303
Non-polymers3,2686
Water0
1


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

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Components

#1: Protein Sodium/nucleoside cotransporter


Mass: 79376.773 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: SLC28A3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: F1MGR1
#2: Chemical ChemComp-XMO / N-hydroxy-5'-O-(2-methylpropanoyl)cytidine / Molnupiravir


Mass: 329.306 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C13H19N3O7 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-LBN / 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / (2R)-2-[(9Z)-9-Octadecenoyloxy]-3-(palmitoyloxy)propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
Has ligand of interestY

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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: bCNT3 trimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.232 MDa / Experimental value: YES
Source (natural)Organism: Bos taurus (cattle)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: sf9
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
1150 mMNaClSodium chloride1
220 mMTris1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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: 81000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.4 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4715
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3particle selection
2Latitude3.5image acquisition
4CTFFIND4CTF correction
7Coot0.96model fitting
9PHENIX1.18model refinement
10cryoSPARC3.3initial Euler assignment
11cryoSPARC3.3final Euler assignment
12cryoSPARC3.3classification
13cryoSPARC3.33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 2592142
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127798 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3TIJ

3tij


Accession code: 3TIJ / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312672
ELECTRON MICROSCOPYf_angle_d0.53517241
ELECTRON MICROSCOPYf_dihedral_angle_d13.8374263
ELECTRON MICROSCOPYf_chiral_restr0.0381989
ELECTRON MICROSCOPYf_plane_restr0.0032091

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