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- PDB-7ydq: Structure of PfNT1(Y190A)-GFP in complex with GSK4 -

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Basic information

Entry
Database: PDB / ID: 7ydq
TitleStructure of PfNT1(Y190A)-GFP in complex with GSK4
ComponentsNucleoside transporter 1,Green fluorescent protein
KeywordsMEMBRANE PROTEIN / malaria / nucleoside transporter / GSK4 / TRANSPORT PROTEIN
Function / homology
Function and homology information


adenosine transport / purine nucleobase transport / nucleoside transmembrane transporter activity / bioluminescence / generation of precursor metabolites and energy / plasma membrane
Similarity search - Function
Equilibrative nucleoside transporter / MFS transporter superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
Chem-IRX / Green fluorescent protein / Nucleoside transporter 1
Similarity search - Component
Biological speciesPlasmodium falciparum 3D7 (eukaryote)
Aequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.04 Å
AuthorsWang, C. / Yu, L.Y. / Li, J.L. / Ren, R.B. / Deng, D.
Funding support China, 2items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)National Key R&D Program of China 2016YFA0502700 China
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
DepositionJul 4, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 26, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Nucleoside transporter 1,Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)77,3772
Polymers77,0361
Non-polymers3411
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Nucleoside transporter 1,Green fluorescent protein


Mass: 77035.766 Da / Num. of mol.: 1 / Mutation: Y190A
Source method: isolated from a genetically manipulated source
Details: PfNT1(Y190A) fused with GFP the GFP was inserted into PfNT1 between K370 and K371
Source: (gene. exp.) Plasmodium falciparum 3D7 (eukaryote), (gene. exp.) Aequorea victoria (jellyfish)
Gene: PF3D7_1347200, GFP / Cell line (production host): IPLB-Sf-21-AE(Sf9) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8IDM6, UniProt: P42212
#2: Chemical ChemComp-IRX / 5-methyl-N-[2-(2-oxidanylideneazepan-1-yl)ethyl]-2-phenyl-1,3-oxazole-4-carboxamide


Mass: 341.404 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H23N3O3 / Feature type: SUBJECT OF INVESTIGATION
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: nucleoside/nucleobase transporter fusion with GFP / Type: COMPLEX
Details: the cDNA of the GFP was cloned into PfNT1 between K370 and K371
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 47.5 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Plasmodium falciparum 3D7 (eukaryote)36329
21Aequorea victoria (jellyfish)6100
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
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: 1100 nm
Image recordingElectron dose: 52.452 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: NONE
3D reconstructionResolution: 4.04 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 329768 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 171.65 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00314897
ELECTRON MICROSCOPYf_angle_d0.69896636
ELECTRON MICROSCOPYf_chiral_restr0.0425757
ELECTRON MICROSCOPYf_plane_restr0.0059825
ELECTRON MICROSCOPYf_dihedral_angle_d4.3851627

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