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- PDB-8oud: Structure of the human neutral amino acid transporter ASCT2 in co... -

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Basic information

Entry
Database: PDB / ID: 8oud
TitleStructure of the human neutral amino acid transporter ASCT2 in complex with nanobody 469
Components
  • Nanobody 469
  • Neutral amino acid transporter B(0)
KeywordsMEMBRANE PROTEIN / Neutral aminoacid transmembrane transporter activity / ASCT2 / Complex / Nanobody
Function / homology
Function and homology information


glutamine secretion / L-glutamine import across plasma membrane / glutamine transport / L-glutamine transmembrane transporter activity / L-serine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity ...glutamine secretion / L-glutamine import across plasma membrane / glutamine transport / L-glutamine transmembrane transporter activity / L-serine transmembrane transporter activity / ligand-gated channel activity / neutral amino acid transport / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / symporter activity / amino acid transport / antiporter activity / RHOJ GTPase cycle / RHOQ GTPase cycle / protein homotrimerization / RHOH GTPase cycle / transport across blood-brain barrier / RAC3 GTPase cycle / RAC1 GTPase cycle / erythrocyte differentiation / basal plasma membrane / melanosome / virus receptor activity / signaling receptor activity / extracellular exosome / membrane / metal ion binding / 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
ALANINE / CHOLESTEROL HEMISUCCINATE / Neutral amino acid transporter B(0)
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.31 Å
AuthorsCanul-Tec, J. / Reyes, N.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)771965European Union
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Receptor-recognition and antiviral mechanisms of retrovirus-derived human proteins.
Authors: Shashank Khare / Miryam I Villalba / Juan C Canul-Tec / Arantza Balsebre Cajiao / Anand Kumar / Marija Backovic / Felix A Rey / Els Pardon / Jan Steyaert / Camilo Perez / Nicolas Reyes /
Abstract: Human syncytin-1 and suppressyn are cellular proteins of retroviral origin involved in cell-cell fusion events to establish the maternal-fetal interface in the placenta. In cell culture, they ...Human syncytin-1 and suppressyn are cellular proteins of retroviral origin involved in cell-cell fusion events to establish the maternal-fetal interface in the placenta. In cell culture, they restrict infections from members of the largest interference group of vertebrate retroviruses, and are regarded as host immunity factors expressed during development. At the core of the syncytin-1 and suppressyn functions are poorly understood mechanisms to recognize a common cellular receptor, the membrane transporter ASCT2. Here, we present cryo-electron microscopy structures of human ASCT2 in complexes with the receptor-binding domains of syncytin-1 and suppressyn. Despite their evolutionary divergence, the two placental proteins occupy similar positions in ASCT2, and are stabilized by the formation of a hybrid β-sheet or 'clamp' with the receptor. Structural predictions of the receptor-binding domains of extant retroviruses indicate overlapping binding interfaces and clamping sites with ASCT2, revealing a competition mechanism between the placental proteins and the retroviruses. Our work uncovers a common ASCT2 recognition mechanism by a large group of endogenous and disease-causing retroviruses, and provides high-resolution views on how placental human proteins exert morphological and immunological functions.
History
DepositionApr 22, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2024Provider: repository / Type: Initial release
Revision 1.1May 15, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Neutral amino acid transporter B(0)
B: Neutral amino acid transporter B(0)
C: Neutral amino acid transporter B(0)
D: Nanobody 469
E: Nanobody 469
F: Nanobody 469
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,22821
Polymers211,2946
Non-polymers1,93415
Water1,67593
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16230 Å2
ΔGint-144 kcal/mol
Surface area62390 Å2
MethodPISA

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Components

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Protein / Antibody , 2 types, 6 molecules ABCDEF

#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 / Alanine Serine Cysteine Transporter 2


Mass: 56638.902 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A5, ASCT2, M7V1, RDR, RDRC / Cell line (production host): HEK293F / Organ (production host): KIDNEY / Production host: Homo sapiens (human) / References: UniProt: Q15758
#2: Antibody Nanobody 469


Mass: 13792.409 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli K-12 (bacteria)

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Non-polymers , 4 types, 108 molecules

#3: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ALA / ALANINE / Alanine


Type: L-peptide linking / Mass: 89.093 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H7NO2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C31H50O4
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 93 / Source method: isolated from a natural source / Formula: H2O

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of human ASCT2 with Nanobody 69COMPLEX#1-#20MULTIPLE SOURCES
2Alanine Serine Cysteine Transporter 2COMPLEX#11RECOMBINANT
3Nanobody 69Single-domain antibodyCOMPLEX#21RECOMBINANT
Molecular weightValue: 0.2 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCell
22Homo sapiens (human)9606HEK 293F
33Escherichia coli K-12 (bacteria)83333
Buffer solutionpH: 7.4
Details: 25 mM Hepes pH7.4, 100 mM NaCl, 5 mM L-Alanine, 0.05% DDS, 0.01% CHS.
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHepesC8H18N2O4S1
2100 mMsodium chlorideNaClSodium chloride1
35 mML-alanineAlanineC3H7NO21
40.05 %Sucrose laurateC24H44O121
50.01 %Cholesteryl hemisuccinate tris saltC31H50O4C4H11NO31
SpecimenConc.: 8.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 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: 130000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4 sec. / Electron dose: 52.24 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4344
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV / Phase plate: VOLTA PHASE PLATE

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1cryoSPARC3.3.2particle selection
2EPU2image acquisition
4cryoSPARC3.3.2CTF correction
7UCSF Chimera1.16model fitting
9PHENIX1.20.1model refinement
10cryoSPARC3.3.2initial Euler assignment
11cryoSPARC3.3.2final Euler assignment
12cryoSPARC3.3.2classification
13cryoSPARC3.3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1346125
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 441100 / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 77.4 / Protocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6GCT

6gct


Pdb chain-ID: A / Accession code: 6GCT / Chain residue range: 43-489 / Pdb chain residue range: 43-489 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 33.73 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003512240
ELECTRON MICROSCOPYf_angle_d0.545216659
ELECTRON MICROSCOPYf_chiral_restr0.04122040
ELECTRON MICROSCOPYf_plane_restr0.0052070
ELECTRON MICROSCOPYf_dihedral_angle_d5.11781782

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