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

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Basic information

Entry
Database: PDB / ID: 7awn
TitleStructure of the thermostabilized EAAT1 cryst mutant in complex with rubidium and barium 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
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 / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / ligand-gated channel activity / Transport of inorganic cations/anions and amino acids/oligopeptides / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / neutral amino acid transport / 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 / transepithelial transport / intracellular sodium ion homeostasis / neurotransmitter transport / antiporter activity / cellular response to cocaine / glutamate binding / amino acid transport / 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 / response to xenobiotic stimulus / neuron projection / 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 / : / RUBIDIUM ION / 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: 3.92 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

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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)57,2955
Polymers56,5121
Non-polymers7834
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)171,88515
Polymers169,5373
Non-polymers2,34812
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 area9840 Å2
ΔGint-138 kcal/mol
Surface area49290 Å2
MethodPISA
Unit cell
Length a, b, c (Å)124.236, 124.236, 91.220
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
Details: Rubidium, Barium and UCPH101 bound,Rubidium, Barium and UCPH101 bound,Rubidium, Barium and 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-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
#3: Chemical ChemComp-BA / BARIUM ION


Mass: 137.327 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ba
#4: Chemical ChemComp-RB / RUBIDIUM ION


Mass: 85.468 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Rb
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: 3.6 Å3/Da / Density % sol: 65.8 %
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: 0.81491 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 16, 2016
RadiationMonochromator: Channel-cut Si / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.81491 Å / Relative weight: 1
ReflectionResolution: 3.92→46.34 Å / Num. obs: 7276 / % possible obs: 99.6 % / Redundancy: 20.4 % / Biso Wilson estimate: 134 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.118 / Rpim(I) all: 0.027 / Rrim(I) all: 0.121 / Net I/σ(I): 10.9 / Num. measured all: 148478
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
3.92-4.0220.16.268103265140.5471.416.4270.695.8
17.53-46.3416.50.04714378710.0120.04963.995.1

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3.92 Å46.34 Å
Translation3.92 Å46.34 Å

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Processing

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

5llm


Resolution: 3.92→21.85 Å / Cor.coef. Fo:Fc: 0.865 / Cor.coef. Fo:Fc free: 0.824 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.804
RfactorNum. reflection% reflectionSelection details
Rfree0.279 294 5.02 %RANDOM
Rwork0.23 ---
obs0.233 5860 80.8 %-
Displacement parametersBiso max: 275.9 Å2 / Biso mean: 148.16 Å2 / Biso min: 29.17 Å2
Baniso -1Baniso -2Baniso -3
1-7.1761 Å20 Å20 Å2
2--7.1761 Å20 Å2
3----14.3522 Å2
Refine analyzeLuzzati coordinate error obs: 0.63 Å
Refinement stepCycle: final / Resolution: 3.92→21.85 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2973 0 35 0 3008
Biso mean--141.45 --
Num. residues----391
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1044SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes46HARMONIC2
X-RAY DIFFRACTIONt_gen_planes470HARMONIC5
X-RAY DIFFRACTIONt_it3055HARMONIC20
X-RAY DIFFRACTIONt_nbd1SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion427SEMIHARMONIC5
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_contact3757SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d3055HARMONIC20.009
X-RAY DIFFRACTIONt_angle_deg4151HARMONIC21.03
X-RAY DIFFRACTIONt_omega_torsion1.84
X-RAY DIFFRACTIONt_other_torsion21.91
LS refinement shellResolution: 3.92→4.38 Å / Rfactor Rfree error: 0 / Total num. of bins used: 5
RfactorNum. reflection% reflection
Rfree0.2715 44 4.67 %
Rwork0.2198 898 -
all0.222 942 -
obs--45.88 %
Refinement TLS params.Method: refined / Origin x: 45.8926 Å / Origin y: -17.8773 Å / Origin z: 0.6181 Å
111213212223313233
T0.1068 Å20.2055 Å20.0302 Å2-0.1598 Å2-0.0271 Å2---0.4082 Å2
L-0.162 °20.0935 °20.3418 °2-0.162 °2-0.0458 °2--0.5254 °2
S-0.2187 Å °0.1236 Å °-0.2594 Å °0.0873 Å °-0.0441 Å °0.017 Å °-0.2978 Å °-0.1627 Å °0.2628 Å °
Refinement TLS groupSelection details: { A|* }

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