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- PDB-8qrs: ASCT2 protomer in lipid nanodiscs with bound glutamine and Na+ io... -

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Basic information

Entry
Database: PDB / ID: 8qrs
TitleASCT2 protomer in lipid nanodiscs with bound glutamine and Na+ ions in the intermediate outward-facing state (iOFS-up)
ComponentsNeutral amino acid transporter B(0)
KeywordsTRANSPORT PROTEIN / neutral amino acid exchanger amino acid transport system elevator transporter glutamine transport
Function / homology
Function and homology information


glutamine secretion / L-glutamine import across plasma membrane / L-glutamine transmembrane transporter activity / glutamine transport / L-serine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity ...glutamine secretion / L-glutamine import across plasma membrane / L-glutamine transmembrane transporter activity / glutamine transport / L-serine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity / symporter activity / Amino acid transport across the plasma membrane / amino acid transmembrane transporter activity / antiporter activity / RHOJ GTPase cycle / protein homotrimerization / RHOQ GTPase cycle / amino acid transport / RHOH GTPase cycle / RAC3 GTPase cycle / transport across blood-brain barrier / RAC1 GTPase cycle / basal plasma membrane / erythrocyte differentiation / centriolar satellite / melanosome / signaling receptor activity / virus receptor activity / ciliary basal body / extracellular exosome / metal ion binding / membrane / plasma membrane
Similarity search - Function
: / Sodium:dicarboxylate symporter family signature 2. / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter superfamily / Sodium:dicarboxylate symporter family / Sodium:dicarboxylate symporter family signature 1.
Similarity search - Domain/homology
GLUTAMINE / Neutral amino acid transporter B(0)
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsBorowska, A. / Rheinberger, J. / Paulino, C. / Slotboom, D.J.
Funding support Netherlands, 2items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)40.018.016 Netherlands
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis of the obligatory exchange mode of human neutral amino acid transporter ASCT2.
Authors: Anna M Borowska / Maria Gabriella Chiariello / Alisa A Garaeva / Jan Rheinberger / Siewert J Marrink / Cristina Paulino / Dirk J Slotboom /
Abstract: ASCT2 is an obligate exchanger of neutral amino acids, contributing to cellular amino acid homeostasis. ASCT2 belongs to the same family (SLC1) as Excitatory Amino Acid Transporters (EAATs) that ...ASCT2 is an obligate exchanger of neutral amino acids, contributing to cellular amino acid homeostasis. ASCT2 belongs to the same family (SLC1) as Excitatory Amino Acid Transporters (EAATs) that concentrate glutamate in the cytosol. The mechanism that makes ASCT2 an exchanger rather than a concentrator remains enigmatic. Here, we employ cryo-electron microscopy and molecular dynamics simulations to elucidate the structural basis of the exchange mechanism of ASCT2. We establish that ASCT2 binds three Na ions per transported substrate and visits a state that likely acts as checkpoint in preventing Na ion leakage, both features shared with EAATs. However, in contrast to EAATs, ASCT2 retains one Na ion even under Na-depleted conditions. We demonstrate that ASCT2 cannot undergo the structural transition in TM7 that is essential for the concentrative transport cycle of EAATs. This structural rigidity and the high-affinity Na binding site effectively confine ASCT2 to an exchange mode.
History
DepositionOct 9, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Neutral amino acid transporter B(0)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,6142
Polymers57,4681
Non-polymers1461
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Neutral amino acid transporter B(0) / ATB(0) / Baboon M7 virus receptor / RD114/simian type D retrovirus receptor / Sodium-dependent ...ATB(0) / Baboon M7 virus receptor / RD114/simian type D retrovirus receptor / Sodium-dependent neutral amino acid transporter type 2 / Solute carrier family 1 member 5


Mass: 57467.793 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A5, ASCT2, M7V1, RDR, RDRC / Production host: Komagataella pastoris (fungus) / References: UniProt: Q15758
#2: Chemical ChemComp-GLN / GLUTAMINE


Type: L-peptide linking / Mass: 146.144 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C5H10N2O3 / 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: ASCT2 in MSP2N2 lipid nanodiscs with glutamine and Na+ ions
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.0534 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.4 / Details: 20 mM Tris/HCl pH 7.4, 200 mM NaCl, 1 mM gln
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 288 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 FIELD / Nominal magnification: 105000 X / Calibrated magnification: 59809 X / Nominal defocus max: 2900 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.43 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 5 / Num. of real images: 14117
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1crYOLO1.7.6particle selection
2EPU2.1image acquisition
4CTFFIND4.1.14CTF correction
7Coot0.9.6model fitting
8ISOLDE1.6.0model fitting
10RELION3.1.0initial Euler assignment
11RELION3.1.0final Euler assignment
13RELION3.1.03D reconstruction
14PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7639781
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 264256 / Algorithm: BACK PROJECTION
Details: The number of particles is the number of protomers from C3 expanded particles
Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6MPB

6mpb


Accession code: 6MPB / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023175
ELECTRON MICROSCOPYf_angle_d0.4364324
ELECTRON MICROSCOPYf_dihedral_angle_d3.394440
ELECTRON MICROSCOPYf_chiral_restr0.034542
ELECTRON MICROSCOPYf_plane_restr0.005535

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