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- PDB-9d69: Human excitatory amino acid transporter 3 (EAAT3) with bound L-Cy... -

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Basic information

Entry
Database: PDB / ID: 9d69
TitleHuman excitatory amino acid transporter 3 (EAAT3) with bound L-Cysteine in an intermediate outward facing state (slight upper position)
ComponentsExcitatory amino acid transporter 3
KeywordsTRANSPORT PROTEIN / human EAAT3 / Cysteine / iOFS-upper
Function / homology
Function and homology information


D-aspartate transmembrane transport / regulation of protein targeting to membrane / 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 / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / neurotransmitter receptor transport to plasma membrane ...D-aspartate transmembrane transport / regulation of protein targeting to membrane / 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 / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / neurotransmitter receptor transport to plasma membrane / L-glutamate import / response to decreased oxygen levels / : / cellular response to mercury ion / retina layer formation / L-glutamate transmembrane transporter activity / L-glutamate transmembrane transport / L-aspartate transmembrane transport / glutathione biosynthetic process / righting reflex / D-aspartate import across plasma membrane / zinc ion transmembrane transport / L-aspartate transmembrane transporter activity / cellular response to ammonium ion / intracellular glutamate homeostasis / Glutamate Neurotransmitter Release Cycle / L-aspartate import across plasma membrane / cellular response to bisphenol A / grooming behavior / L-glutamate import across plasma membrane / proximal dendrite / monoatomic anion channel activity / intracellular zinc ion homeostasis / transepithelial transport / apical dendrite / blood vessel morphogenesis / chloride transmembrane transporter activity / cellular response to cocaine / motor neuron apoptotic process / G protein-coupled dopamine receptor signaling pathway / response to anesthetic / superoxide metabolic process / glutamate receptor signaling pathway / response to morphine / heart contraction / perisynaptic space / neurotransmitter transport / maintenance of blood-brain barrier / dopamine metabolic process / motor behavior / adult behavior / asymmetric synapse / conditioned place preference / glial cell projection / response to axon injury / postsynaptic modulation of chemical synaptic transmission / positive regulation of heart rate / transport across blood-brain barrier / behavioral fear response / synaptic cleft / monoatomic ion transport / neurogenesis / axon terminus / chloride transmembrane transport / dendritic shaft / cell periphery / response to amphetamine / locomotory behavior / brain development / synapse organization / recycling endosome / Schaffer collateral - CA1 synapse / recycling endosome membrane / cytokine-mediated signaling pathway / memory / apical part of cell / long-term synaptic potentiation / late endosome membrane / presynapse / cellular response to oxidative stress / early endosome membrane / gene expression / dendritic spine / chemical synaptic transmission / negative regulation of neuron apoptotic process / perikaryon / apical plasma membrane / membrane raft / response to xenobiotic stimulus / 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
CYSTEINE / : / Excitatory amino acid transporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.99 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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,5126
Polymers57,1211
Non-polymers3915
Water00
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.: 1
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-CYS / CYSTEINE


Type: L-peptide linking / Mass: 121.158 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C3H7NO2S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / 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.: 1 / Source method: obtained synthetically / Formula: Hg / Feature type: SUBJECT OF INVESTIGATION
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 monomer with L-Cys 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 L-Cysteine
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
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: 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: 2400 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 58.25 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
1RELIONparticle selection
4CTFFINDCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
12RELION3classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4180263
Details: This is the number of trimer particle from the image
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60670
Details: This is number of monomer from C3 expanded particles.
Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 59.17 / Protocol: OTHER
Atomic model buildingPDB-ID: 8CV3

8cv3


Accession code: 8CV3 / Source name: PDB / Type: experimental model

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