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- PDB-6ruq: Structure of GluA2cryst in complex the antagonist ZK200775 and th... -

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Basic information

Entry
Database: PDB / ID: 6ruq
TitleStructure of GluA2cryst in complex the antagonist ZK200775 and the negative allosteric modulator GYKI53655 at 4.65 A resolution
ComponentsGlutamate receptor 2GRIA2
KeywordsMEMBRANE PROTEIN / Receptor Negative allosteric modulator Antagonist Non-competitve antagonist
Function / homology
Function and homology information


spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / response to lithium ion / perisynaptic space / cellular response to glycine / AMPA glutamate receptor activity / Trafficking of GluR2-containing AMPA receptors / immunoglobulin binding ...spine synapse / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / response to lithium ion / perisynaptic space / cellular response to glycine / AMPA glutamate receptor activity / Trafficking of GluR2-containing AMPA receptors / immunoglobulin binding / AMPA glutamate receptor complex / kainate selective glutamate receptor activity / ionotropic glutamate receptor complex / extracellularly glutamate-gated ion channel activity / asymmetric synapse / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / glutamate receptor binding / positive regulation of synaptic transmission / glutamate-gated receptor activity / presynaptic active zone membrane / response to fungicide / regulation of synaptic transmission, glutamatergic / cellular response to brain-derived neurotrophic factor stimulus / somatodendritic compartment / dendrite membrane / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / ionotropic glutamate receptor binding / ionotropic glutamate receptor signaling pathway / dendrite cytoplasm / cytoskeletal protein binding / SNARE binding / dendritic shaft / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic membrane / synaptic transmission, glutamatergic / PDZ domain binding / postsynaptic density membrane / protein tetramerization / modulation of chemical synaptic transmission / Schaffer collateral - CA1 synapse / establishment of protein localization / terminal bouton / receptor internalization / synaptic vesicle membrane / cerebral cortex development / synaptic vesicle / presynapse / signaling receptor activity / presynaptic membrane / amyloid-beta binding / growth cone / perikaryon / chemical synaptic transmission / postsynaptic membrane / scaffold protein binding / dendritic spine / postsynaptic density / neuron projection / axon / dendrite / neuronal cell body / glutamatergic synapse / synapse / protein-containing complex binding / endoplasmic reticulum membrane / protein kinase binding / cell surface / endoplasmic reticulum / protein-containing complex / membrane / identical protein binding / plasma membrane
Similarity search - Function
Bacterial extracellular solute-binding proteins, family 3 / Solute-binding protein family 3/N-terminal domain of MltF / 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. ...Bacterial extracellular solute-binding proteins, family 3 / Solute-binding protein family 3/N-terminal domain of MltF / 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
Chem-GYK / Chem-ZK1 / Glutamate receptor 2
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 4.65 Å
AuthorsKrintel, C. / Venskutonyte, R. / Mirza, O.A. / Gajhede, M. / Kastrup, J.S.
Funding support Denmark, 5items
OrganizationGrant numberCountry
Danish Council for Independent Research Denmark
Other government Denmark
Lundbeckfonden Denmark
Other government Denmark
Other government Denmark
CitationJournal: FEBS J / Year: 2021
Title: Binding of a negative allosteric modulator and competitive antagonist can occur simultaneously at the ionotropic glutamate receptor GluA2.
Authors: Christian Krintel / Jerzy Dorosz / Andreas Haahr Larsen / Thor Seneca Thorsen / Raminta Venskutonytė / Osman Mirza / Michael Gajhede / Thomas Boesen / Jette Sandholm Kastrup /
Abstract: Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: ...Ionotropic glutamate receptors are ligand-gated ion channels governing neurotransmission in the central nervous system. Three major types of antagonists are known for the AMPA-type receptor GluA2: competitive, noncompetitive (i.e., negative allosteric modulators; NAMs) used for treatment of epilepsy, and uncompetitive antagonists. We here report a 4.65 Å resolution X-ray structure of GluA2, revealing that four molecules of the competitive antagonist ZK200775 and four molecules of the NAM GYKI53655 are capable of binding at the same time. Using negative stain electron microscopy, we show that GYKI53655 alone or ZK200775/GYKI53655 in combination predominantly results in compact receptor forms. The agonist AMPA provides a mixed population of compact and bulgy shapes of GluA2 not impacted by addition of GYKI53655. Taken together, this suggests that the two different mechanisms of antagonism that lead to channel closure are independent and that the distribution between bulgy and compact receptors primarily depends on the ligand bound in the glutamate binding site. DATABASE: The atomic coordinates and structure factors from the crystal structure determination have been deposited in the Protein Data Bank under accession code https://doi.org/10.2210/pdb6RUQ/pdb. The electron microscopy 3D reconstruction volumes have been deposited in EMDB (EMD-4875: Apo; EMD-4920: ZK200775/GYKI53655; EMD-4921: AMPA compact; EMD-4922: AMPA/GYKI53655 bulgy; EMD-4923: GYKI53655; EMD-4924: AMPA bulgy; EMD-4925: AMPA/GYKI53655 compact).
History
DepositionMay 28, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 24, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 1, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / audit_author ...atom_site / audit_author / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.pdbx_formal_charge / _atom_site.type_symbol / _audit_author.name / _chem_comp.name / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.src_method / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Mar 3, 2021Group: Database references / Structure summary / Category: chem_comp / citation / citation_author
Item: _chem_comp.pdbx_synonyms / _citation.journal_volume ..._chem_comp.pdbx_synonyms / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 2.2Jan 24, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)373,08415
Polymers368,1164
Non-polymers4,96811
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area30950 Å2
ΔGint-192 kcal/mol
Surface area140950 Å2
MethodPISA
Unit cell
Length a, b, c (Å)91.773, 306.596, 107.728
Angle α, β, γ (deg.)90.000, 94.264, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
21
31
41

NCS domain segments:

Ens-ID: 1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ASNASNARGARG(chain 'A' and (resid 10 through 545 or resid 572 through 816))AA10 - 5451 - 536
12ALAALACYSCYS(chain 'A' and (resid 10 through 545 or resid 572 through 816))AA572 - 815563 - 806
23ASNASNARGARGchain 'B'BB10 - 5451 - 536
24ALAALACYSCYSchain 'B'BB572 - 815563 - 806
35ASNASNARGARG(chain 'C' and (resid 10 through 545 or resid 572 through 816))CC10 - 5451 - 536
36ALAALACYSCYS(chain 'C' and (resid 10 through 545 or resid 572 through 816))CC572 - 815563 - 806
47ASNASNARGARG(chain 'D' and (resid 10 through 545 or resid 572 through 816))DD10 - 5451 - 536
48ALAALACYSCYS(chain 'D' and (resid 10 through 545 or resid 572 through 816))DD572 - 815563 - 806

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Components

#1: Protein
Glutamate receptor 2 / GRIA2 / 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: 92029.008 Da / Num. of mol.: 4 / Mutation: N235E, N385D, N392Q, K410A, R586Q, C589A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Production host: Spodoptera (butterflies/moths) / References: UniProt: P19491
#2: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#3: Polysaccharide beta-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4) ...beta-D-mannopyranose-(1-3)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-3DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2-2/a4-b1_b4-c1_c3-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][b-D-Manp]{}}}}}LINUCSPDB-CARE
#4: Chemical
ChemComp-GYK / (8R)-5-(4-aminophenyl)-N,8-dimethyl-8,9-dihydro-2H,7H-[1,3]dioxolo[4,5-h][2,3]benzodiazepine-7-carboxamide


Mass: 352.387 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C19H20N4O3 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ZK1 / {[7-morpholin-4-yl-2,3-dioxo-6-(trifluoromethyl)-3,4-dihydroquinoxalin-1(2H)-yl]methyl}phosphonic acid / [[3,4-Dihydro-7-(4-morpholinyl)-2,3-dioxo-6-(trifluorom ethyl)-1(2H)-quinoxalinyl]methyl]phosphonic acid / Fanapanel


Mass: 409.254 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H15F3N3O6P / Feature type: SUBJECT OF INVESTIGATION / Comment: antagonist, medication*YM

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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: 4.11 Å3/Da / Density % sol: 70.04 %
Crystal growTemperature: 279 K / Method: microbatch / pH: 6 / Details: 0.1 M MES, 12 % PEG 20,000

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 0.95373 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Jul 19, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.95373 Å / Relative weight: 1
ReflectionResolution: 4.65→53.71 Å / Num. obs: 31560 / % possible obs: 99.1 % / Redundancy: 5.6 % / Biso Wilson estimate: 167.32 Å2 / CC1/2: 0.991 / Net I/σ(I): 6.8
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
4.65-4.775.71.0321331323170.3130.4611.1351.999.6
20.8-53.715.60.04520083570.9850.0220.05129.294.8

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
MOSFLMdata reduction
Aimless0.2.14data scaling
PHASERphasing
PHENIXrefinement
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5L1H

5l1h


Resolution: 4.65→51.1 Å / SU ML: 0.8322 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 33.8346
RfactorNum. reflection% reflection
Rfree0.2914 1559 4.95 %
Rwork0.2424 --
obs0.2448 31505 99.04 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 210.87 Å2
Refinement stepCycle: LAST / Resolution: 4.65→51.1 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms24710 0 340 0 25050
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.002625607
X-RAY DIFFRACTIONf_angle_d0.583634664
X-RAY DIFFRACTIONf_chiral_restr0.04083846
X-RAY DIFFRACTIONf_plane_restr0.00524348
X-RAY DIFFRACTIONf_dihedral_angle_d6.389615084
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
4.65-4.80.35131550.3092704X-RAY DIFFRACTION99.44
4.8-4.970.33971570.29982675X-RAY DIFFRACTION99.51
4.97-5.170.34951130.29062791X-RAY DIFFRACTION99.28
5.17-5.410.37711280.28952731X-RAY DIFFRACTION98.52
5.41-5.690.36071520.27352698X-RAY DIFFRACTION98.41
5.69-6.050.32881460.25612692X-RAY DIFFRACTION99.4
6.05-6.510.30111410.2312737X-RAY DIFFRACTION99.45
6.51-7.170.28551420.21422737X-RAY DIFFRACTION99.65
7.17-8.20.25041340.19822720X-RAY DIFFRACTION98.45
8.2-10.320.21081610.18882714X-RAY DIFFRACTION99.1
10.32-51.10.29011300.25162747X-RAY DIFFRACTION98.33

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