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- EMDB-17189: Structure of the human neutral amino acid transporter ASCT2 in co... -

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Basic information

Entry
Database: EMDB / ID: EMD-17189
TitleStructure of the human neutral amino acid transporter ASCT2 in complex with nanobody 469
Map data
Sample
  • Complex: Complex of human ASCT2 with Nanobody 69
    • Complex: Alanine Serine Cysteine Transporter 2
      • Protein or peptide: Neutral amino acid transporter B(0)
    • Complex: Nanobody 69Single-domain antibody
      • Protein or peptide: Nanobody 469
  • Ligand: SODIUM IONSodium
  • Ligand: ALANINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water
KeywordsNeutral aminoacid transmembrane transporter activity / ASCT2 / Complex / Nanobody / MEMBRANE PROTEIN
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 / basal plasma membrane / erythrocyte differentiation / 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
Neutral amino acid transporter B(0)
Similarity search - Component
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.31 Å
AuthorsCanul-Tec J / Reyes N
Funding supportEuropean Union, 1 items
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, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17189.png

Downloads & links

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Map

FileDownload / File: emd_17189.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 280 pix.
= 261.52 Å		0.93 Å/pix.
x 280 pix.
= 261.52 Å		0.93 Å/pix.
x 280 pix.
= 261.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.934 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-1.6338122 - 2.562065
Average (Standard dev.)0.0015460027 (±0.055972785)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 261.52002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_17189_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_17189_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of human ASCT2 with Nanobody 69

EntireName: Complex of human ASCT2 with Nanobody 69
Components
  • Complex: Complex of human ASCT2 with Nanobody 69
    • Complex: Alanine Serine Cysteine Transporter 2
      • Protein or peptide: Neutral amino acid transporter B(0)
    • Complex: Nanobody 69Single-domain antibody
      • Protein or peptide: Nanobody 469
  • Ligand: SODIUM IONSodium
  • Ligand: ALANINE
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: water

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Supramolecule #1: Complex of human ASCT2 with Nanobody 69

SupramoleculeName: Complex of human ASCT2 with Nanobody 69 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Molecular weightTheoretical: 200 KDa

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Supramolecule #2: Alanine Serine Cysteine Transporter 2

SupramoleculeName: Alanine Serine Cysteine Transporter 2 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Nanobody 69

SupramoleculeName: Nanobody 69 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Neutral amino acid transporter B(0)

MacromoleculeName: Neutral amino acid transporter B(0) / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 56.638902 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MVADPPRDSK GLAAAEPTAN GGLALASIED QGAAAGGYCG SRDQVRRCLR ANLLVLLTVV AVVAGVALGL GVSGAGGALA LGPERLSAF VFPGELLLRL LRMIILPLVV CSLIGGAASL DPGALGRLGA WALLFFLVTT LLASALGVGL ALALQPGAAS A AINASVGA ...String:
MVADPPRDSK GLAAAEPTAN GGLALASIED QGAAAGGYCG SRDQVRRCLR ANLLVLLTVV AVVAGVALGL GVSGAGGALA LGPERLSAF VFPGELLLRL LRMIILPLVV CSLIGGAASL DPGALGRLGA WALLFFLVTT LLASALGVGL ALALQPGAAS A AINASVGA AGSAENAPSK EVLDSFLDLA RNIFPSNLVS AAFRSYSTTY EERNITGTRV KVPVGQEVEG MNILGLVVFA IV FGVALRK LGPEGELLIR FFNSFNEATM VLVSWIMWYA PVGIMFLVAG KIVEMEDVGL LFARLGKYIL CCLLGHAIHG LLV LPLIYF LFTRKNPYRF LWGIVTPLAT AFGTSSSSAT LPLMMKCVEE NNGVAKHISR FILPIGATVN MDGAALFQCV AAVF IAQLS QQSLDFVKII TILVTATASS VGAAGIPAGG VLTLAIILEA VNLPVDHISL ILAVDWLVDR SCTVLNVEGD ALGAG LLQN YVDRTESRST EPELIQVKSE LPLDPLPVPT EEGNPLLKHY RGPAGDATVA SEKESVM

UniProtKB: Neutral amino acid transporter B(0)

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Macromolecule #2: Nanobody 469

MacromoleculeName: Nanobody 469 / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 13.792409 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString:
QVQLVESGGG LVQPGGSLRL SCAASGSIFR LDAMGWYRQA PGKQRELVAV IRSGGSTDYG DSVKGRFTIS RDNAKNTVYL QMNSLKPED TAVYYCNAVQ ILKTIYWGQG TQVTVSSHHH HHHEPEA

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 9
Molecular weightTheoretical: 22.99 Da

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Macromolecule #4: ALANINE

MacromoleculeName: ALANINE / type: ligand / ID: 4 / Number of copies: 3 / Formula: ALA
Molecular weightTheoretical: 89.093 Da
Chemical component information

ChemComp-ALA:
ALANINE / Alanine

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 3 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 93 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration8.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHepes
100.0 mMNaClSodium chloridesodium chloride
5.0 mMC3H7NO2L-alanineAlanine
0.05 %C24H44O12Sucrose laurate
0.01 %C31H50O4C4H11NO3Cholesteryl hemisuccinate tris salt

Details: 25 mM Hepes pH7.4, 100 mM NaCl, 5 mM L-Alanine, 0.05% DDS, 0.01% CHS.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 40 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4344 / Average exposure time: 4.0 sec. / Average electron dose: 52.24 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1346125
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6gct

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 38 / Software - Name: cryoSPARC (ver. 3.3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.2)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.31 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 441100
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6gct


Chain - Chain ID: A / Chain - Residue range: 43-489 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 77.4
Output model

PDB-8oud:
Structure of the human neutral amino acid transporter ASCT2 in complex with nanobody 469

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