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- PDB-9d67: Human excitatory amino acid transporter 3 (EAAT3) with bound D-As... -

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Basic information

Entry
Database: PDB / ID: 9d67
TitleHuman excitatory amino acid transporter 3 (EAAT3) with bound D-Aspartate in an intermediate outward facing state
ComponentsExcitatory amino acid transporter 3
KeywordsTRANSPORT PROTEIN / human EAAT3 / D-Asp / iOFS
Function / homology
Function and homology information


D-aspartate transmembrane transport / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / L-cysteine transport / L-cysteine transmembrane transporter activity / L-glutamate import / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / SLC-mediated transport of amino acids ...D-aspartate transmembrane transport / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / L-cysteine transport / L-cysteine transmembrane transporter activity / L-glutamate import / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / SLC-mediated transport of amino acids / L-glutamate transmembrane transporter activity / glutathione biosynthetic process / grooming behavior / L-glutamate transmembrane transport / L-aspartate transmembrane transport / zinc ion transmembrane transport / L-aspartate transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-aspartate import across plasma membrane / D-aspartate import across plasma membrane / blood vessel morphogenesis / monoatomic anion channel activity / proximal dendrite / L-glutamate import across plasma membrane / intracellular zinc ion homeostasis / motor behavior / transepithelial transport / cellular response to cocaine / apical dendrite / chloride transmembrane transporter activity / G protein-coupled dopamine receptor signaling pathway / superoxide metabolic process / adult behavior / response to morphine / maintenance of blood-brain barrier / perisynaptic space / dopamine metabolic process / retina development in camera-type eye / glutamate receptor signaling pathway / neurotransmitter transport / amino acid transport / asymmetric synapse / glial cell projection / postsynaptic modulation of chemical synaptic transmission / conditioned place preference / positive regulation of heart rate / neurogenesis / synaptic cleft / transport across blood-brain barrier / response to amphetamine / axon terminus / chloride transmembrane transport / locomotory behavior / cell periphery / dendritic shaft / brain development / memory / synapse organization / recycling endosome / neuron apoptotic process / Schaffer collateral - CA1 synapse / gene expression / recycling endosome membrane / cytokine-mediated signaling pathway / apical part of cell / late endosome membrane / presynapse / chemical synaptic transmission / early endosome membrane / negative regulation of neuron apoptotic process / dendritic spine / perikaryon / apical plasma membrane / membrane raft / axon / neuronal cell body / synapse / dendrite / cell surface / extracellular exosome / membrane / metal ion binding / identical protein 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
D-ASPARTIC ACID / : / Excitatory amino acid transporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsQiu, B. / Boudker, O.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS085318 United States
CitationJournal: bioRxiv / Year: 2024
Title: Structural basis of the excitatory amino acid transporter 3 substrate recognition.
Authors: Biao Qiu / Olga Boudker /
Abstract: Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only ...Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent work suggests that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate promiscuity by determining the cryo-EM structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures illustrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.
History
DepositionAug 14, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Excitatory amino acid transporter 3
B: Excitatory amino acid transporter 3
C: Excitatory amino acid transporter 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)172,57118
Polymers171,3633
Non-polymers1,20815
Water543
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Excitatory amino acid transporter 3 / Excitatory amino-acid carrier 1 / Neuronal and epithelial glutamate transporter / Sodium-dependent ...Excitatory amino-acid carrier 1 / Neuronal and epithelial glutamate transporter / Sodium-dependent glutamate/aspartate transporter 3 / Solute carrier family 1 member 1


Mass: 57120.863 Da / Num. of mol.: 3
Mutation: C9A, C100A, C158A, N178T, N195T, C219A, C256W, K269C, W441C.
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A1, EAAC1, EAAT3 / Production host: Homo sapiens (human) / References: UniProt: P43005
#2: Chemical ChemComp-DAS / D-ASPARTIC ACID


Type: D-peptide linking / Mass: 133.103 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
#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-HG / MERCURY (II) ION


Mass: 200.590 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Hg / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: EAAT3 with D-Asp bound at iOFS* / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Details: 20 mM Hepes-Tris pH 7.4, 200 mM NaCl and 0.01% GDN, 10 mM D-Aspartate
SpecimenConc.: 5 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
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM300FEG/T
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 52.19 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
4RELIONCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
12cryoSPARC3classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3346010
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 391308 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 65.75 / Protocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 8CTC

8ctc


Accession code: 8CTC / Source name: PDB / Type: experimental model

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