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- PDB-7awq: Structure of the thermostabilized EAAT1 cryst-E386Q mutant in com... -

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Basic information

Entry
Database: PDB / ID: 7awq
TitleStructure of the thermostabilized EAAT1 cryst-E386Q mutant in complex with L-ASP, 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 / ion-coupling mechanism / TRANSPORT PROTEIN
Function / homology
Function and homology information


Defective SLC1A3 causes episodic ataxia 6 (EA6) / auditory behavior / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / glutamine secretion / neurotransmitter uptake / L-glutamine import across plasma membrane / cranial nerve development / cell morphogenesis involved in neuron differentiation / glutamine transport ...Defective SLC1A3 causes episodic ataxia 6 (EA6) / auditory behavior / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / glutamine secretion / neurotransmitter uptake / L-glutamine import across plasma membrane / cranial nerve development / cell morphogenesis involved in neuron differentiation / glutamine transport / L-glutamine transmembrane transporter activity / L-serine transmembrane transporter activity / gamma-aminobutyric acid biosynthetic process / ligand-gated channel activity / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / Transport of inorganic cations/anions and amino acids/oligopeptides / neutral amino acid transport / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / D-aspartate import across plasma membrane / amino acid transmembrane transporter activity / Amino acid transport across the plasma membrane / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / symporter activity / intracellular sodium ion homeostasis / transepithelial transport / neurotransmitter transport / antiporter activity / amino acid transport / cellular response to cocaine / glutamate binding / RHOJ GTPase cycle / RHOQ GTPase cycle / protein homotrimerization / RHOH GTPase cycle / neuromuscular process controlling balance / transport across blood-brain barrier / response to light stimulus / RAC3 GTPase cycle / positive regulation of synaptic transmission / monoatomic ion transport / potassium ion transmembrane transport / chloride transmembrane transport / RAC1 GTPase cycle / erythrocyte differentiation / basal plasma membrane / sensory perception of sound / response to wounding / melanosome / virus receptor activity / signaling 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: 3.65 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)57,2506
Polymers56,5111
Non-polymers7395
Water0
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)171,75118
Polymers169,5343
Non-polymers2,21715
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_545-y,x-y-1,z1
crystal symmetry operation3_655-x+y+1,-x,z1
Buried area8560 Å2
ΔGint-118 kcal/mol
Surface area52510 Å2
MethodPISA
Unit cell
Length a, b, c (Å)124.000, 124.000, 90.810
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: 56511.395 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ...Details: L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound,L-ASP, sodium ions and allosteric inhibitor UCPH101 bound
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.: 2 / Source method: obtained synthetically / Formula: Na
#3: Chemical ChemComp-ASP / ASPARTIC ACID / 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: 3.57 Å3/Da / Density % sol: 65.5 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8.2
Details: 30% PEG400, 100 mM 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: ESRF / Beamline: ID29 / Wavelength: 0.9793 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Nov 13, 2018
RadiationMonochromator: Channel-cut Si / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 3.65→28 Å / Num. obs: 8923 / % possible obs: 99.9 % / Redundancy: 16.925 % / Biso Wilson estimate: 115.6 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.144 / Rrim(I) all: 0.149 / Χ2: 0.719 / Net I/σ(I): 8.5
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
3.65-3.7414.4214.710.576440.4234.885100
3.74-3.8516.9053.1960.966240.5793.295100
3.85-3.9617.1351.9861.686300.8442.046100
3.96-4.0817.871.3862.476010.9411.426100
4.08-4.2117.6641.1242.915870.9511.157100
4.21-4.3617.3510.7444.225610.9680.767100
4.36-4.5316.8830.5625.465540.9750.58100
4.53-4.7116.6350.4276.545210.9860.44100
4.71-4.9217.1070.3437.715160.9920.354100
4.92-5.1617.7480.3297.954800.9950.339100
5.16-5.4417.7430.3158.244630.9940.32599.8
5.44-5.7717.4050.3238.314420.9790.333100
5.77-6.1716.4750.2769.624130.9860.285100
6.17-6.6616.9840.22711.893870.9860.234100
6.66-7.317.5670.16716.73580.9940.172100
7.3-8.1617.2970.13923.053200.9950.143100
8.16-9.4215.570.10127.792790.9980.105100
9.42-11.5416.7850.09530.992470.9970.098100
11.54-16.3216.1010.09131.11890.9990.09499.5
16.32-2814.430.08231.241070.9970.08595.5

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3.6 Å46.21 Å
Translation3.6 Å46.21 Å

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Processing

Software
NameVersionClassification
BUSTERrefinement
XDSJan 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: 3.65→28 Å / Cor.coef. Fo:Fc: 0.757 / Cor.coef. Fo:Fc free: 0.67 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.638
RfactorNum. reflection% reflectionSelection details
Rfree0.271 382 5.02 %RANDOM
Rwork0.233 ---
obs0.235 7604 85.4 %-
Displacement parametersBiso max: 260.36 Å2 / Biso mean: 96.1 Å2 / Biso min: 9.55 Å2
Baniso -1Baniso -2Baniso -3
1-4.7146 Å20 Å20 Å2
2--4.7146 Å20 Å2
3----9.4293 Å2
Refine analyzeLuzzati coordinate error obs: 0.55 Å
Refinement stepCycle: final / Resolution: 3.65→28 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3074 0 44 0 3118
Biso mean--80.7 --
Num. residues----404
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1091SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes54HARMONIC2
X-RAY DIFFRACTIONt_gen_planes482HARMONIC5
X-RAY DIFFRACTIONt_it3162HARMONIC20
X-RAY DIFFRACTIONt_nbd3SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion443SEMIHARMONIC5
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_contact3841SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d3162HARMONIC20.009
X-RAY DIFFRACTIONt_angle_deg4292HARMONIC21.03
X-RAY DIFFRACTIONt_omega_torsion1.87
X-RAY DIFFRACTIONt_other_torsion21.42
LS refinement shellResolution: 3.65→4.08 Å / Rfactor Rfree error: 0 / Total num. of bins used: 5
RfactorNum. reflection% reflection
Rfree0.2274 74 5.71 %
Rwork0.2031 1222 -
all0.2045 1296 -
obs--51.95 %
Refinement TLS params.

L33: 0 °2 / Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.32570.0214-0.0933-0.08390.0769-0.1253-0.0167-0.0165-0.0519-0.0821-0.2117-0.2275-0.37740.20740.0246-0.09710.03490.243-0.0062-0.404442.424-37.15925.8007
21.215-0.37230.0939-0.79410.7425-0.03940.16230.01360.0101-0.0015-0.05110.0998-0.12340.041-0.27610.1873-0.03660.2097-0.0745-0.41532.6864-24.43497.2489
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{A|29 - 111 A|170 - 283}A29 - 111
2X-RAY DIFFRACTION1{A|29 - 111 A|170 - 283}A170 - 283
3X-RAY DIFFRACTION2{A|112 - 147 A|290 - 488}A112 - 147
4X-RAY DIFFRACTION2{A|112 - 147 A|290 - 488}A290 - 488

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