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- PDB-8vj7: GluA2 bound to GYKI-52466 and Glutamate, Inhibited State 2 -

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Basic information

Entry
Database: PDB / ID: 8vj7
TitleGluA2 bound to GYKI-52466 and Glutamate, Inhibited State 2
ComponentsIsoform Flip of Glutamate receptor 2
KeywordsMEMBRANE PROTEIN/INHIBITOR / ligand gated ion channel / ionotropic glutamate receptor / allosteric inhibition / MEMBRANE PROTEIN / MEMBRANE PROTEIN-INHIBITOR complex
Function / homology
Function and homology information


spine synapse / dendritic spine cytoplasm / dendritic spine neck / cellular response to amine stimulus / dendritic spine head / Activation of AMPA receptors / ligand-gated monoatomic cation channel activity / perisynaptic space / Trafficking of GluR2-containing AMPA receptors / AMPA glutamate receptor activity ...spine synapse / dendritic spine cytoplasm / dendritic spine neck / cellular response to amine stimulus / dendritic spine head / Activation of AMPA receptors / ligand-gated monoatomic cation channel activity / perisynaptic space / Trafficking of GluR2-containing AMPA receptors / AMPA glutamate receptor activity / response to lithium ion / AMPA glutamate receptor clustering / kainate selective glutamate receptor activity / immunoglobulin binding / asymmetric synapse / AMPA glutamate receptor complex / regulation of receptor recycling / extracellularly glutamate-gated ion channel activity / cellular response to glycine / ionotropic glutamate receptor complex / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / conditioned place preference / positive regulation of synaptic transmission / response to fungicide / regulation of synaptic transmission, glutamatergic / extracellular ligand-gated monoatomic ion channel activity / glutamate-gated receptor activity / cytoskeletal protein binding / cellular response to brain-derived neurotrophic factor stimulus / regulation of long-term synaptic depression / somatodendritic compartment / glutamate-gated calcium ion channel activity / presynaptic active zone membrane / excitatory synapse / ionotropic glutamate receptor signaling pathway / ionotropic glutamate receptor binding / dendrite membrane / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / dendrite cytoplasm / positive regulation of excitatory postsynaptic potential / dendritic shaft / SNARE binding / synaptic membrane / PDZ domain binding / synaptic transmission, glutamatergic / establishment of protein localization / protein tetramerization / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / cerebral cortex development / receptor internalization / postsynaptic density membrane / modulation of chemical synaptic transmission / Schaffer collateral - CA1 synapse / terminal bouton / synaptic vesicle / long-term synaptic potentiation / synaptic vesicle membrane / amyloid-beta binding / growth cone / presynapse / signaling receptor activity / presynaptic membrane / scaffold protein binding / dendritic spine / chemical synaptic transmission / perikaryon / postsynaptic membrane / neuron projection / postsynaptic density / external side of plasma membrane / axon / neuronal cell body / synapse / dendrite / protein kinase binding / protein-containing complex binding / glutamatergic synapse / cell surface / endoplasmic reticulum / protein-containing complex / membrane / identical protein binding / plasma membrane
Similarity search - Function
Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / : / Ionotropic glutamate receptor / Eukaryotic homologues of bacterial periplasmic substrate binding proteins. / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I
Similarity search - Domain/homology
: / GLUTAMIC ACID / Glutamate receptor 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.85 Å
AuthorsHale, W.D. / Montano Romero, A. / Huganir, R.L. / Twomey, E.C.
Funding support United States, 5items
OrganizationGrant numberCountry
Other privateKinship Foundation 22098168
Other privateDiana Helis Henry Medical Research Foundation 142548
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH112152 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS036715 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH132811 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Allosteric competition and inhibition in AMPA receptors.
Authors: W Dylan Hale / Alejandra Montaño Romero / Cuauhtemoc U Gonzalez / Vasanthi Jayaraman / Albert Y Lau / Richard L Huganir / Edward C Twomey /
Abstract: Excitatory neurotransmission is principally mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype ionotropic glutamate receptors (AMPARs). Negative allosteric modulators ...Excitatory neurotransmission is principally mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype ionotropic glutamate receptors (AMPARs). Negative allosteric modulators are therapeutic candidates that inhibit AMPAR activation and can compete with positive modulators to control AMPAR function through unresolved mechanisms. Here we show that allosteric inhibition pushes AMPARs into a distinct state that prevents both activation and positive allosteric modulation. We used cryo-electron microscopy to capture AMPARs bound to glutamate, while a negative allosteric modulator, GYKI-52466, and positive allosteric modulator, cyclothiazide, compete for control of the AMPARs. GYKI-52466 binds in the ion channel collar and inhibits AMPARs by decoupling the ligand-binding domains from the ion channel. The rearrangement of the ligand-binding domains ruptures the cyclothiazide site, preventing positive modulation. Our data provide a framework for understanding allostery of AMPARs and for rational design of therapeutics targeting AMPARs in neurological diseases.
History
DepositionJan 5, 2024Deposition site: RCSB / Processing site: RCSB
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform Flip of Glutamate receptor 2
B: Isoform Flip of Glutamate receptor 2
C: Isoform Flip of Glutamate receptor 2
D: Isoform Flip of Glutamate receptor 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)358,69412
Polymers356,9324
Non-polymers1,7628
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
Isoform Flip of Glutamate receptor 2 / GluR-2 / AMPA-selective glutamate receptor 2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / ...GluR-2 / AMPA-selective glutamate receptor 2 / GluR-B / GluR-K2 / Glutamate receptor ionotropic / AMPA 2 / GluA2


Mass: 89232.961 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Cell line (production host): HEK 293 Gnti- / Production host: Homo sapiens (human) / References: UniProt: P19491
#2: Chemical
ChemComp-GLU / GLUTAMIC ACID


Type: L-peptide linking / Mass: 147.129 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C5H9NO4
#3: Chemical
ChemComp-A1AB5 / 4-[(5S,8R)-8-methyl-6,7,8,9-tetrahydro-2H,5H-[1,3]dioxolo[4,5-h][2,3]benzodiazepin-5-yl]aniline


Mass: 293.320 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C17H15N3O2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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: AMPA Receptor GluA2 Homotetramer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293 Gnti-
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 2600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameVersionCategory
2EPU3.51image acquisition
4cryoSPARCCTF correction
8PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130474 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00431150
ELECTRON MICROSCOPYf_angle_d0.58142074
ELECTRON MICROSCOPYf_dihedral_angle_d7.6764221
ELECTRON MICROSCOPYf_chiral_restr0.0414664
ELECTRON MICROSCOPYf_plane_restr0.0045283

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