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- PDB-7awm: Structure of the thermostabilized EAAT1 cryst mutant in complex w... -

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Basic information

Entry
Database: PDB / ID: 7awm
TitleStructure of the thermostabilized EAAT1 cryst mutant in complex with L-ASP, three sodium ions and the allosteric inhibitor UCPH101
ComponentsExcitatory amino acid transporter 1,Neutral amino acid transporter B(0),Excitatory amino acid transporter 1
KeywordsMEMBRANE PROTEIN / EXCITATORY AMINO ACID TRANSPORTER 1 / HUMAN GLUTAMATE TRANSPORTER / SLC1A3 / allosteric inhibitor UCPH101
Function / homology
Function and homology information


Defective SLC1A3 causes episodic ataxia 6 (EA6) / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / auditory behavior / neurotransmitter uptake / cranial nerve development / cell morphogenesis involved in neuron differentiation / glutamine secretion / gamma-aminobutyric acid biosynthetic process / high-affinity L-glutamate transmembrane transporter activity ...Defective SLC1A3 causes episodic ataxia 6 (EA6) / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / auditory behavior / neurotransmitter uptake / cranial nerve development / cell morphogenesis involved in neuron differentiation / glutamine secretion / gamma-aminobutyric acid biosynthetic process / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamine import across plasma membrane / L-glutamate import / L-glutamine transmembrane transporter activity / glutamine transport / L-serine transmembrane transporter activity / Transport of inorganic cations/anions and amino acids/oligopeptides / ligand-gated channel activity / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / D-aspartate import across plasma membrane / neutral amino acid transport / amino acid transmembrane transporter activity / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / Glutamate Neurotransmitter Release Cycle / Amino acid transport across the plasma membrane / neutral L-amino acid transmembrane transporter activity / L-glutamate import across plasma membrane / transepithelial transport / symporter activity / intracellular sodium ion homeostasis / cellular response to cocaine / neurotransmitter transport / antiporter activity / glutamate binding / amino acid transport / RHOJ GTPase cycle / RHOQ GTPase cycle / protein homotrimerization / neuromuscular process controlling balance / RHOH GTPase cycle / transport across blood-brain barrier / response to light stimulus / RAC3 GTPase cycle / positive regulation of synaptic transmission / monoatomic ion transport / chloride transmembrane transport / potassium ion transmembrane transport / RAC1 GTPase cycle / basal plasma membrane / erythrocyte differentiation / sensory perception of sound / response to wounding / melanosome / signaling receptor activity / virus receptor activity / cytoplasmic vesicle / chemical synaptic transmission / neuron projection / response to xenobiotic stimulus / response to antibiotic / neuronal cell body / synapse / perinuclear region of cytoplasm / cell surface / 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
Chem-6Z6 / ASPARTIC ACID / : / Excitatory amino acid transporter 1 / Neutral amino acid transporter B(0)
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.25 Å
AuthorsCanul-Tec, J.C. / Legrand, P. / Reyes, N.
Funding support France, 1items
OrganizationGrant numberCountry
European Research Council (ERC)309657 France
CitationJournal: EMBO J / Year: 2022
Title: The ion-coupling mechanism of human excitatory amino acid transporters.
Authors: Juan C Canul-Tec / Anand Kumar / Jonathan Dhenin / Reda Assal / Pierre Legrand / Martial Rey / Julia Chamot-Rooke / Nicolas Reyes /
Abstract: Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co- ...Excitatory amino acid transporters (EAATs) maintain glutamate gradients in the brain essential for neurotransmission and to prevent neuronal death. They use ionic gradients as energy source and co-transport transmitter into the cytoplasm with Na and H , while counter-transporting K to re-initiate the transport cycle. However, the molecular mechanisms underlying ion-coupled transport remain incompletely understood. Here, we present 3D X-ray crystallographic and cryo-EM structures, as well as thermodynamic analysis of human EAAT1 in different ion bound conformations, including elusive counter-transport ion bound states. Binding energies of Na and H , and unexpectedly Ca , are coupled to neurotransmitter binding. Ca competes for a conserved Na site, suggesting a regulatory role for Ca in glutamate transport at the synapse, while H binds to a conserved glutamate residue stabilizing substrate occlusion. The counter-transported ion binding site overlaps with that of glutamate, revealing the K -based mechanism to exclude the transmitter during the transport cycle and to prevent its neurotoxic release on the extracellular side.
History
DepositionNov 8, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 13, 2021Provider: repository / Type: Initial release
Revision 1.1Dec 1, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 12, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID
Revision 1.3Jan 19, 2022Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Excitatory amino acid transporter 1,Neutral amino acid transporter B(0),Excitatory amino acid transporter 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,2747
Polymers56,5121
Non-polymers7626
Water00
1
A: Excitatory amino acid transporter 1,Neutral amino acid transporter B(0),Excitatory amino acid transporter 1
hetero molecules

A: Excitatory amino acid transporter 1,Neutral amino acid transporter B(0),Excitatory amino acid transporter 1
hetero molecules

A: Excitatory amino acid transporter 1,Neutral amino acid transporter B(0),Excitatory amino acid transporter 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,82321
Polymers169,5373
Non-polymers2,28618
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-y,x-y+1,z1
crystal symmetry operation3_455-x+y-1,-x,z1
Buried area9220 Å2
ΔGint-156 kcal/mol
Surface area52430 Å2
MethodPISA
Unit cell
Length a, b, c (Å)122.882, 122.882, 90.032
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number173
Space group name H-MP63

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Components

#1: Protein Excitatory amino acid transporter 1,Neutral amino acid transporter B(0),Excitatory amino acid transporter 1 / Sodium-dependent glutamate/aspartate transporter 1 / GLAST-1 / Solute carrier family 1 member 3 / ...Sodium-dependent glutamate/aspartate transporter 1 / GLAST-1 / Solute carrier family 1 member 3 / 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 / Sodium-dependent glutamate/aspartate transporter 1 / GLAST-1 / Solute carrier family 1 member 3


Mass: 56512.379 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: SLC1A3, EAAT1, GLAST, GLAST1, SLC1A5, ASCT2, M7V1, RDR, RDRC
Plasmid: pcDNA3.1 / Cell (production host): embrionic / Cell line (production host): HEK-293F / Production host: Homo sapiens (human) / Tissue (production host): embrionic kidney / References: UniProt: P43003, UniProt: Q15758
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na
#3: Chemical ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H7NO4
#4: Chemical ChemComp-6Z6 / 2-Amino-5,6,7,8-tetrahydro-4-(4-methoxyphenyl)-7-(naphthalen-1-yl)-5-oxo-4H-chromene-3-carbonitrile


Mass: 422.475 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H22N2O3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-BA / BARIUM ION


Mass: 137.327 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ba
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.47 Å3/Da / Density % sol: 64.58 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8.2
Details: 28% PEG400, 100mM Tris pH 8.2, 50 mM Calcium chloride, 50 mM Barium chloride
PH range: 8-8.4

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.979 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Sep 23, 2016
RadiationMonochromator: Channel cut monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.979 Å / Relative weight: 1
ReflectionResolution: 3.25→45.81 Å / Num. obs: 12293 / % possible obs: 99.8 % / Redundancy: 13.5 % / Biso Wilson estimate: 145 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.146 / Rpim(I) all: 0.042 / Rrim(I) all: 0.152 / Net I/σ(I): 8.8 / Num. measured all: 166319
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. measured allNum. unique obsCC1/2Rpim(I) allRrim(I) allNet I/σ(I) obs% possible all
3.25-3.3312.57.927111298870.1752.2828.2640.498.1
14.53-45.8110.80.0716141500.9980.0240.07434.896.5

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3.25 Å45.02 Å
Translation3.25 Å45.02 Å

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
XDSJune 31, 2020 Built=20200417data reduction
Aimless0.5.32data scaling
PHASER2.7.17phasing
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5LLM

5llm


Resolution: 3.25→23.31 Å / Cor.coef. Fo:Fc: 0.912 / Cor.coef. Fo:Fc free: 0.894 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.458
RfactorNum. reflection% reflectionSelection details
Rfree0.238 440 4.55 %RANDOM
Rwork0.219 ---
obs0.22 9675 78.9 %-
Displacement parametersBiso max: 265.43 Å2 / Biso mean: 135.42 Å2 / Biso min: 75.45 Å2
Baniso -1Baniso -2Baniso -3
1-4.682 Å20 Å20 Å2
2--4.682 Å20 Å2
3----9.3641 Å2
Refine analyzeLuzzati coordinate error obs: 0.48 Å
Refinement stepCycle: final / Resolution: 3.25→23.31 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3135 0 45 0 3180
Biso mean--113.18 --
Num. residues----412
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1110SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes56HARMONIC2
X-RAY DIFFRACTIONt_gen_planes493HARMONIC5
X-RAY DIFFRACTIONt_it3227HARMONIC20
X-RAY DIFFRACTIONt_nbd2SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion448SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact4022SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d3227HARMONIC20.01
X-RAY DIFFRACTIONt_angle_deg4382HARMONIC21.17
X-RAY DIFFRACTIONt_omega_torsion2.19
X-RAY DIFFRACTIONt_other_torsion22.47
LS refinement shellResolution: 3.25→3.63 Å / Rfactor Rfree error: 0 / Total num. of bins used: 5
RfactorNum. reflection% reflection
Rfree0.2183 56 4.58 %
Rwork0.2169 1166 -
all0.217 1222 -
obs--35.23 %
Refinement TLS params.Method: refined / Origin x: -36.0755 Å / Origin y: 30.1168 Å / Origin z: 4.5558 Å
111213212223313233
T-0.2676 Å20.1246 Å2-0.002 Å2-0.1714 Å20.0733 Å2---0.39 Å2
L2.4757 °2-0.0831 °2-0.0382 °2-1.7581 °2-0.0988 °2--3.5616 °2
S-0.0792 Å °-0.1292 Å °-0.298 Å °0.0956 Å °-0.0713 Å °-0.5971 Å °0.4311 Å °1.2529 Å °0.1505 Å °
Refinement TLS groupSelection details: { A|* }

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