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- PDB-7awl: Structure of the thermostabilized EAAT1 cryst-II mutant in comple... -

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Basic information

Entry
Database: PDB / ID: 7awl
TitleStructure of the thermostabilized EAAT1 cryst-II mutant in complex with barium and the allosteric inhibitor UCPH101
ComponentsExcitatory amino acid transporter 1, Neutral amino acid transporter, Excitatory amino acid transporter, Transporter protein
KeywordsMEMBRANE PROTEIN / excitatory amino acid transporter 1 / human glutamate transporter / SLC1A3 / ion-coupling mechanism
Function / homologyChem-6Z6 / :
Function and homology information
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.7 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 / _citation_author.identifier_ORCID / _citation_author.name
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, Excitatory amino acid transporter, Transporter protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)57,1574
Polymers56,4601
Non-polymers6973
Water00
1
A: Excitatory amino acid transporter 1, Neutral amino acid transporter, Excitatory amino acid transporter, Transporter protein
hetero molecules

A: Excitatory amino acid transporter 1, Neutral amino acid transporter, Excitatory amino acid transporter, Transporter protein
hetero molecules

A: Excitatory amino acid transporter 1, Neutral amino acid transporter, Excitatory amino acid transporter, Transporter protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,47212
Polymers169,3813
Non-polymers2,0919
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 area8590 Å2
ΔGint-108 kcal/mol
Surface area47700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)124.940, 124.940, 92.020
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, Excitatory amino acid transporter, Transporter protein


Mass: 56460.324 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Barium and inhibitor UCPH101 / Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A3, EAAT1, GLAST / Plasmid: pcDNA3.1 / Cell (production host): embrionic / Cell line (production host): HEK-293F / Production host: Homo sapiens (human) / Tissue (production host): embrionic kidney
#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
#3: Chemical ChemComp-BA / BARIUM ION


Mass: 137.327 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ba / Feature type: SUBJECT OF INVESTIGATION
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: 3.67 Å3/Da / Density % sol: 66.5 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 8.2
Details: 32% 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: SOLEIL / Beamline: PROXIMA 1 / Wavelength: 0.9786 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: 0.9786 Å / Relative weight: 1
ReflectionResolution: 3.7→22 Å / Num. obs: 8827 / % possible obs: 99.9 % / Redundancy: 20.546 % / Biso Wilson estimate: 120.5 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.125 / Rrim(I) all: 0.129 / Χ2: 0.823 / Net I/σ(I): 11.76 / Num. measured all: 181357 / Scaling rejects: 172
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
3.7-3.820.3016.7850.55133386586570.4016.95899.8
3.8-3.920.1584.2270.93127406336320.6484.33799.8
3.9-4.0121.0472.2231.92125655985970.8732.27899.8
4.01-4.1420.8891.7192.36126386056050.9211.761100
4.14-4.2720.4551.2683118645805800.9251.3100
4.27-4.4221.30.9134.37120135655640.9570.93599.8
4.42-4.5921.0810.5816.2114265425420.9860.595100
4.59-4.7820.1140.4427.1100575005000.9880.454100
4.78-4.9920.8440.3299.01108395205200.9960.337100
4.99-5.2320.3640.2988.9796124744720.9970.30599.6
5.23-5.5220.9760.2739.9195654564560.9980.28100
5.52-5.8521.1140.26911.6390374284280.9970.276100
5.85-6.2619.9490.23912.4182194124120.9950.245100
6.26-6.7621.0180.1718.3481133863860.9980.174100
6.76-7.420.260.11425.570103463460.9970.117100
7.4-8.2820.9170.07837.7965683143140.9980.08100
8.28-9.5619.8180.06744.6256482852850.9990.069100
9.56-11.719.5270.06346.9846282372370.9970.064100
11.7-16.5519.4740.06247.9337001901900.9990.064100
16.55-2217.0870.06847.1117771101040.9980.07194.5

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation3.7 Å46.64 Å
Translation3.7 Å46.64 Å

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Processing

Software
NameVersionClassification
XDSJan 31, 2020 Built=20200417data reduction
XSCALEJan 31, 2020 Buit=2020417data scaling
PHASER2.7.17phasing
BUSTER2.10.3refinement
PDB_EXTRACT3.27data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5LM4

5lm4


Resolution: 3.7→22 Å / Cor.coef. Fo:Fc: 0.89 / Cor.coef. Fo:Fc free: 0.851 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.753
RfactorNum. reflection% reflectionSelection details
Rfree0.285 319 4.89 %RANDOM
Rwork0.237 ---
obs0.24 6520 74.2 %-
Displacement parametersBiso max: 288.76 Å2 / Biso mean: 164.86 Å2 / Biso min: 54.56 Å2
Baniso -1Baniso -2Baniso -3
1-8.6227 Å20 Å20 Å2
2--8.6227 Å20 Å2
3----17.2454 Å2
Refine analyzeLuzzati coordinate error obs: 0.61 Å
Refinement stepCycle: final / Resolution: 3.7→22 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2953 0 34 0 2987
Biso mean--157.77 --
Num. residues----389
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d1036SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes44HARMONIC2
X-RAY DIFFRACTIONt_gen_planes467HARMONIC5
X-RAY DIFFRACTIONt_it3034HARMONIC20
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_contact3785SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d3034HARMONIC20.009
X-RAY DIFFRACTIONt_angle_deg4124HARMONIC21.04
X-RAY DIFFRACTIONt_omega_torsion1.95
X-RAY DIFFRACTIONt_other_torsion21.55
LS refinement shellResolution: 3.7→4.14 Å / Rfactor Rfree error: 0 / Total num. of bins used: 5
RfactorNum. reflection% reflection
Rfree0.2275 46 5.11 %
Rwork0.22 855 -
all0.2203 901 -
obs--36.18 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°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)
1-0.54480.31450.19620.54480.23730-0.2117-0.06470.0247-0.12120.1553-0.3204-0.2189-0.37840.05640.1607-0.03730.00840.4180.086-0.446845.1987-38.03641.5606
22.54860.0603-2.3772-0.0852-0.46962.1089-0.0910.1671-0.1674-0.0469-0.47230.0029-0.61140.20550.5633-0.39620.1107-0.03250.5290.0421-0.448633.3603-26.85952.0839
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{ A|42 - A|111 A|170 - A|283 }A42 - 111
2X-RAY DIFFRACTION1{ A|42 - A|111 A|170 - A|283 }A170 - 283
3X-RAY DIFFRACTION2{ A|112 - A|147 A|295 - A|486 }A112 - 147
4X-RAY DIFFRACTION2{ A|112 - A|147 A|295 - A|486 }A295 - 486

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