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- PDB-7wn0: Structure of PfENT1(Y190A) in complex with nanobody 19 -

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Basic information

Entry
Database: PDB / ID: 7wn0
TitleStructure of PfENT1(Y190A) in complex with nanobody 19
Components
  • Equilibrative nucleoside/nucleobase transporter
  • nanobody19
KeywordsTRANSPORT PROTEIN / malaria / nucleoside transporter
Function / homologyEquilibrative nucleoside transporter / nucleoside transmembrane transporter activity / MFS transporter superfamily / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta / plasma membrane / Equilibrative nucleoside/nucleobase transporter
Function and homology information
Biological speciesPlasmodium falciparum (malaria parasite P. falciparum)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.64 Å
AuthorsWang, C. / Deng, D. / Ren, R.B. / Yu, L.Y.
Funding support China, 2items
OrganizationGrant numberCountry
Other government2016YFA0502700
National Natural Science Foundation of China (NSFC)32171211 China
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of the substrate recognition and inhibition mechanism of Plasmodium falciparum nucleoside transporter PfENT1.
Authors: Chen Wang / Leiye Yu / Jiying Zhang / Yanxia Zhou / Bo Sun / Qingjie Xiao / Minhua Zhang / Huayi Liu / Jinhong Li / Jialu Li / Yunzi Luo / Jie Xu / Zhong Lian / Jingwen Lin / Xiang Wang / ...Authors: Chen Wang / Leiye Yu / Jiying Zhang / Yanxia Zhou / Bo Sun / Qingjie Xiao / Minhua Zhang / Huayi Liu / Jinhong Li / Jialu Li / Yunzi Luo / Jie Xu / Zhong Lian / Jingwen Lin / Xiang Wang / Peng Zhang / Li Guo / Ruobing Ren / Dong Deng /
Abstract: By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates ...By lacking de novo purine biosynthesis enzymes, Plasmodium falciparum requires purine nucleoside uptake from host cells. The indispensable nucleoside transporter ENT1 of P. falciparum facilitates nucleoside uptake in the asexual blood stage. Specific inhibitors of PfENT1 prevent the proliferation of P. falciparum at submicromolar concentrations. However, the substrate recognition and inhibitory mechanism of PfENT1 are still elusive. Here, we report cryo-EM structures of PfENT1 in apo, inosine-bound, and inhibitor-bound states. Together with in vitro binding and uptake assays, we identify that inosine is the primary substrate of PfENT1 and that the inosine-binding site is located in the central cavity of PfENT1. The endofacial inhibitor GSK4 occupies the orthosteric site of PfENT1 and explores the allosteric site to block the conformational change of PfENT1. Furthermore, we propose a general "rocker switch" alternating access cycle for ENT transporters. Understanding the substrate recognition and inhibitory mechanisms of PfENT1 will greatly facilitate future efforts in the rational design of antimalarial drugs.
History
DepositionJan 17, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 1, 2023Provider: repository / Type: Initial release
Revision 1.0Feb 1, 2023Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 1, 2023Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 1, 2023Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 1, 2023Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 1, 2023Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Feb 1, 2023Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 1, 2023Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
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
Revision 1.2Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update
Revision 1.3Jul 2, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jul 2, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Equilibrative nucleoside/nucleobase transporter
B: nanobody19


Theoretical massNumber of molelcules
Total (without water)60,7602
Polymers60,7602
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Equilibrative nucleoside/nucleobase transporter


Mass: 47579.801 Da / Num. of mol.: 1 / Mutation: Y190A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Plasmodium falciparum (malaria parasite P. falciparum)
Gene: NT1 / Cell line (production host): IPLB-Sf-21-AE(sf9) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NIH7
#2: Antibody nanobody19


Mass: 13180.519 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Bacillus subtilis (bacteria)
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1PfENT1(Y190A) in complex with nanobody 19COMPLEXall0MULTIPLE SOURCES
2PfENT1COMPLEX#11RECOMBINANT
3nanobody 19COMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Plasmodium falciparum (malaria parasite P. falciparum)5833
32Vicugna pacos (alpaca)30538
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Spodoptera frugiperda (fall armyworm)7108
32Bacillus subtilis (bacteria)1423
Buffer solutionpH: 6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 1800 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 57.5 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89264 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033971
ELECTRON MICROSCOPYf_angle_d0.5555391
ELECTRON MICROSCOPYf_dihedral_angle_d4.209519
ELECTRON MICROSCOPYf_chiral_restr0.041628
ELECTRON MICROSCOPYf_plane_restr0.005663

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