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- EMDB-33408: Structure of human excitatory amino acid transporter 2 (EAAT2) in... -

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Basic information

Entry
Database: EMDB / ID: EMD-33408
TitleStructure of human excitatory amino acid transporter 2 (EAAT2) in complex with WAY-213613
Map dataEAAT2_WAY213.mrc
Sample
  • Complex: transporterTransport protein
    • Protein or peptide: Excitatory amino acid transporter 2
  • Ligand: (2S)-2-azanyl-4-[[4-[2-bromanyl-4,5-bis(fluoranyl)phenoxy]phenyl]amino]-4-oxidanylidene-butanoic acid
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: dodecyl beta-D-glucopyranoside
Function / homology
Function and homology information


neurotransmitter reuptake / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / cysteine transmembrane transporter activity / high-affinity L-glutamate transmembrane transporter activity / visual behavior / glutamate:sodium symporter activity / Transport of inorganic cations/anions and amino acids/oligopeptides / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity ...neurotransmitter reuptake / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / cysteine transmembrane transporter activity / high-affinity L-glutamate transmembrane transporter activity / visual behavior / glutamate:sodium symporter activity / Transport of inorganic cations/anions and amino acids/oligopeptides / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / L-aspartate transmembrane transport / D-aspartate import across plasma membrane / glutathione biosynthetic process / : / telencephalon development / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity / monoatomic anion transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / astrocyte projection / transepithelial transport / neuron projection terminus / neurotransmitter transport / cellular response to cocaine / adult behavior / protein homotrimerization / transport across blood-brain barrier / axolemma / response to amino acid / monoatomic ion transport / positive regulation of glucose import / multicellular organism growth / response to wounding / presynaptic membrane / cell body / chemical synaptic transmission / vesicle / response to xenobiotic stimulus / membrane raft / glutamatergic synapse / cell surface / 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
Excitatory amino acid transporter 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsZhao Y / Zhang Z
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC) China
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis of ligand binding modes of human EAAT2.
Authors: Zhenglai Zhang / Huiwen Chen / Ze Geng / Zhuoya Yu / Hang Li / Yanli Dong / Hongwei Zhang / Zhuo Huang / Juquan Jiang / Yan Zhao /
Abstract: In the central nervous system (CNS), excitatory amino acid transporters (EAATs) mediate the uptake of excitatory neurotransmitter glutamate and maintain its low concentrations in the synaptic cleft ...In the central nervous system (CNS), excitatory amino acid transporters (EAATs) mediate the uptake of excitatory neurotransmitter glutamate and maintain its low concentrations in the synaptic cleft for avoiding neuronal cytotoxicity. Dysfunction of EAATs can lead to many psychiatric diseases. Here we report cryo-EM structures of human EAAT2 in an inward-facing conformation, in the presence of substrate glutamate or selective inhibitor WAY-213613. The glutamate is coordinated by extensive hydrogen bonds and further stabilized by HP2. The inhibitor WAY-213613 occupies a similar binding pocket to that of the substrate glutamate. Upon association with the WAY-213613, the HP2 undergoes a substantial conformational change, and in turn stabilizes the inhibitor binding by forming hydrophobic interactions. Electrophysiological experiments elucidate that the unique S441 plays pivotal roles in the binding of hEAAT2 with glutamate or WAY-213613, and the I464-L467-V468 cluster acts as a key structural determinant for the selective inhibition of this transporter by WAY-213613.
History
DepositionMay 9, 2022-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33408.png

Downloads & links

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Map

FileDownload / File: emd_33408.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEAAT2_WAY213.mrc
Voxel sizeX=Y=Z: 1.04 Å
Density
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-2.4803536 - 3.797376
Average (Standard dev.)0.0008985505 (±0.08982089)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 266.24 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: map half A.mrc

Fileemd_33408_half_map_1.map
Annotationmap_half_A.mrc
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: map half B.mrc

Fileemd_33408_half_map_2.map
Annotationmap_half_B.mrc
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : transporter

EntireName: transporterTransport protein
Components
  • Complex: transporterTransport protein
    • Protein or peptide: Excitatory amino acid transporter 2
  • Ligand: (2S)-2-azanyl-4-[[4-[2-bromanyl-4,5-bis(fluoranyl)phenoxy]phenyl]amino]-4-oxidanylidene-butanoic acid
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: dodecyl beta-D-glucopyranoside

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Supramolecule #1: transporter

SupramoleculeName: transporter / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Excitatory amino acid transporter 2

MacromoleculeName: Excitatory amino acid transporter 2 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 62.164977 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MASTEGANNM PKQVEVRMHD SHLGSEEPKH RHLGLRLCDK LGKNLLLTLT VFGVILGAVC GGLLRLASPI HPDVVMLIAF PGDILMRML KMLILPLIIS SLITGLSGLD AKASGRLGTR AMVYYMSTTI IAAVLGVILV LAIHPGNPKL KKQLGPGKKN D EVSSLDAF ...String:
MASTEGANNM PKQVEVRMHD SHLGSEEPKH RHLGLRLCDK LGKNLLLTLT VFGVILGAVC GGLLRLASPI HPDVVMLIAF PGDILMRML KMLILPLIIS SLITGLSGLD AKASGRLGTR AMVYYMSTTI IAAVLGVILV LAIHPGNPKL KKQLGPGKKN D EVSSLDAF LDLIRNLFPE NLVQACFQQI QTVTKKVLVA PPPDEEANAT SAVVSLLNET VTEVPEETKM VIKKGLEFKD GM NVLGLIG FFIAFGIAMG KMGDQAKLMV DFFNILNEIV MKLVIMIMWY SPLGIACLIC GKIIAIKDLE VVARQLGMYM VTV IIGLII HGGIFLPLIY FVVTRKNPFS FFAGIFQAWI TALGTASSAG TLPVTFRCLE ENLGIDKRVT RFVLPVGATI NMDG TALYE AVAAIFIAQM NGVVLDGGQI VTVSLTATLA SVGAASIPSA GLVTMLLILT AVGLPTEDIS LLVAVDWLLD RMRTS VNVV GDSFGAGIVY HLSKSELDTI DSQHRVHEDI EMTKTQSIYD DMKNHRESNS NQCVYAAHNS VIVDECKVTL AANGKS ADC SVEEEPWKRE K

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Macromolecule #2: (2S)-2-azanyl-4-[[4-[2-bromanyl-4,5-bis(fluoranyl)phenoxy]phenyl]...

MacromoleculeName: (2S)-2-azanyl-4-[[4-[2-bromanyl-4,5-bis(fluoranyl)phenoxy]phenyl]amino]-4-oxidanylidene-butanoic acid
type: ligand / ID: 2 / Number of copies: 3 / Formula: GJ0
Molecular weightTheoretical: 415.186 Da
Chemical component information

ChemComp-GJ0:
(2S)-2-azanyl-4-[[4-[2-bromanyl-4,5-bis(fluoranyl)phenoxy]phenyl]amino]-4-oxidanylidene-butanoic acid / inhibitor*YM / WAY-213,613

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Macromolecule #3: CHOLESTEROL HEMISUCCINATE

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

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #4: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 4 / Number of copies: 30 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM / Phosphatidylcholine

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Macromolecule #5: dodecyl beta-D-glucopyranoside

MacromoleculeName: dodecyl beta-D-glucopyranoside / type: ligand / ID: 5 / Number of copies: 3 / Formula: XKJ
Molecular weightTheoretical: 348.475 Da
Chemical component information

ChemComp-XKJ:
dodecyl beta-D-glucopyranoside / detergent*YM / Lauryl glucoside

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 420

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