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- PDB-8bsu: Crystal structure of the kainate receptor GluK3-H523A ligand bind... -

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Basic information

Entry
Database: PDB / ID: 8bsu
TitleCrystal structure of the kainate receptor GluK3-H523A ligand binding domain in complex with kainate and the positive allosteric modulator BPAM344 at 2.9A resolution
ComponentsGlutamate receptor ionotropic, kainate 3
KeywordsMEMBRANE PROTEIN / IONOTROPIC GLUTAMATE RECEPTOR / GLUK3 LIGAND-BINDING DOMAIN / GluK3-H523A-LBD / positive allosteric modulator / BPAM344
Function / homology
Function and homology information


Presynaptic function of Kainate receptors / regulation of presynaptic membrane potential / cochlear hair cell ribbon synapse / adenylate cyclase inhibiting G protein-coupled glutamate receptor activity / kainate selective glutamate receptor complex / G protein-coupled glutamate receptor signaling pathway / Activation of Ca-permeable Kainate Receptor / negative regulation of synaptic transmission, glutamatergic / glutamate receptor activity / glutamate receptor signaling pathway ...Presynaptic function of Kainate receptors / regulation of presynaptic membrane potential / cochlear hair cell ribbon synapse / adenylate cyclase inhibiting G protein-coupled glutamate receptor activity / kainate selective glutamate receptor complex / G protein-coupled glutamate receptor signaling pathway / Activation of Ca-permeable Kainate Receptor / negative regulation of synaptic transmission, glutamatergic / glutamate receptor activity / glutamate receptor signaling pathway / kainate selective glutamate receptor activity / glutamate-gated receptor activity / ligand-gated monoatomic ion channel activity involved in regulation of presynaptic membrane potential / dendrite cytoplasm / regulation of membrane potential / synaptic transmission, glutamatergic / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / postsynaptic density membrane / modulation of chemical synaptic transmission / terminal bouton / presynaptic membrane / monoatomic ion transmembrane transport / chemical synaptic transmission / perikaryon / postsynaptic membrane / axon / glutamatergic synapse / dendrite / plasma membrane
Similarity search - Function
Ionotropic glutamate receptor, metazoa / Ligated ion channel L-glutamate- and glycine-binding site / Ionotropic glutamate receptor, L-glutamate and glycine-binding domain / Ligated ion channel L-glutamate- and glycine-binding site / Ligand-gated ion channel / : / 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-2J9 / ACETATE ION / 3-(CARBOXYMETHYL)-4-ISOPROPENYLPROLINE / Glutamate receptor ionotropic, kainate 3
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.9 Å
AuthorsVenskutonyte, R. / Frydenvang, K. / Kastrup, J.S.
Funding support1items
OrganizationGrant numberCountry
Not funded
Citation
Journal: FEBS J / Year: 2024
Title: Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.
Authors: Yasmin Bay / Raminta Venskutonytė / Stine M Frantsen / Thor S Thorsen / Maria Musgaard / Karla Frydenvang / Pierre Francotte / Bernard Pirotte / Philip C Biggin / Anders S Kristensen / ...Authors: Yasmin Bay / Raminta Venskutonytė / Stine M Frantsen / Thor S Thorsen / Maria Musgaard / Karla Frydenvang / Pierre Francotte / Bernard Pirotte / Philip C Biggin / Anders S Kristensen / Thomas Boesen / Darryl S Pickering / Michael Gajhede / Jette S Kastrup /
Abstract: The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in ...The kainate receptors GluK1-3 (glutamate receptor ionotropic, kainate receptors 1-3) belong to the family of ionotropic glutamate receptors and are essential for fast excitatory neurotransmission in the brain, and are associated with neurological and psychiatric diseases. How these receptors can be modulated by small-molecule agents is not well understood, especially for GluK3. We show that the positive allosteric modulator BPAM344 can be used to establish robust calcium-sensitive fluorescence-based assays to test agonists, antagonists, and positive allosteric modulators of GluK1-3. The half-maximal effective concentration (EC) of BPAM344 for potentiating the response of 100 μm kainate was determined to be 26.3 μm for GluK1, 75.4 μm for GluK2, and 639 μm for GluK3. Domoate was found to be a potent agonist for GluK1 and GluK2, with an EC of 0.77 and 1.33 μm, respectively, upon co-application of 150 μm BPAM344. At GluK3, domoate acts as a very weak agonist or antagonist with a half-maximal inhibitory concentration (IC) of 14.5 μm, in presence of 500 μm BPAM344 and 100 μm kainate for competition binding. Using H523A-mutated GluK3, we determined the first dimeric structure of the ligand-binding domain by X-ray crystallography, allowing location of BPAM344, as well as zinc-, sodium-, and chloride-ion binding sites at the dimer interface. Molecular dynamics simulations support the stability of the ion sites as well as the involvement of Asp761, Asp790, and Glu797 in the binding of zinc ions. Using electron microscopy, we show that, in presence of glutamate and BPAM344, full-length GluK3 adopts a dimer-of-dimers arrangement.
#1: Journal: Febs J. / Year: 2024
Title: Small-molecule positive allosteric modulation of homomeric kainate receptors GluK1-3: development of screening assays and insight into GluK3 structure.
Authors: Bay, Y. / Venskutonyte, R. / Frantsen, S.M. / Thorsen, T.S. / Musgaard, M. / Frydenvang, K. / Francotte, P. / Pirotte, B. / Biggin, P.C. / Kristensen, A.S. / Boesen, T. / Pickering, D.S. / ...Authors: Bay, Y. / Venskutonyte, R. / Frantsen, S.M. / Thorsen, T.S. / Musgaard, M. / Frydenvang, K. / Francotte, P. / Pirotte, B. / Biggin, P.C. / Kristensen, A.S. / Boesen, T. / Pickering, D.S. / Gajhede, M. / Kastrup, J.S.
History
DepositionNov 26, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Jul 3, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate receptor ionotropic, kainate 3
B: Glutamate receptor ionotropic, kainate 3
C: Glutamate receptor ionotropic, kainate 3
D: Glutamate receptor ionotropic, kainate 3
E: Glutamate receptor ionotropic, kainate 3
F: Glutamate receptor ionotropic, kainate 3
G: Glutamate receptor ionotropic, kainate 3
H: Glutamate receptor ionotropic, kainate 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)240,07890
Polymers232,2038
Non-polymers7,87582
Water2,072115
1
A: Glutamate receptor ionotropic, kainate 3
D: Glutamate receptor ionotropic, kainate 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,04921
Polymers58,0512
Non-polymers1,99919
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Glutamate receptor ionotropic, kainate 3
C: Glutamate receptor ionotropic, kainate 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,93823
Polymers58,0512
Non-polymers1,88821
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
E: Glutamate receptor ionotropic, kainate 3
H: Glutamate receptor ionotropic, kainate 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)60,46427
Polymers58,0512
Non-polymers2,41325
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
F: Glutamate receptor ionotropic, kainate 3
G: Glutamate receptor ionotropic, kainate 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)59,62719
Polymers58,0512
Non-polymers1,57617
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)83.145, 100.559, 132.826
Angle α, β, γ (deg.)90.00, 103.93, 90.00
Int Tables number4
Space group name H-MP1211

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Components

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Protein , 1 types, 8 molecules ABCDEFGH

#1: Protein
Glutamate receptor ionotropic, kainate 3 / GluK3 / Glutamate receptor 7 / GluR-7 / GluR7


Mass: 29025.383 Da / Num. of mol.: 8 / Mutation: H523A
Source method: isolated from a genetically manipulated source
Details: THE PROTEIN CRYSTALLIZED IS THE EXTRACELLULAR LIGAND BINDING DOMAIN OF GLUK3-H523A. TRANSMEMBRANE REGIONS WERE GENETICALLY REMOVED AND REPLACED WITH A GLY-THR LINKER (RESIDUES 119 AND 120 OF ...Details: THE PROTEIN CRYSTALLIZED IS THE EXTRACELLULAR LIGAND BINDING DOMAIN OF GLUK3-H523A. TRANSMEMBRANE REGIONS WERE GENETICALLY REMOVED AND REPLACED WITH A GLY-THR LINKER (RESIDUES 119 AND 120 OF THE STRUCTURE). THEREFORE, THE SEQUENCE MATCHES DISCONTINUOUSLY WITH THE REFERENCE DATABASE (432-546, 669-806)
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Grik3, Glur7 / Plasmid: PLASMID / Details (production host): POPINJ / Production host: Escherichia coli (E. coli) / Strain (production host): ORIGAMI 2 / References: UniProt: P42264

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Non-polymers , 8 types, 197 molecules

#2: Chemical
ChemComp-2J9 / 4-cyclopropyl-7-fluoro-3,4-dihydro-2H-1,2,4-benzothiadiazine 1,1-dioxide


Mass: 242.270 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H11FN2O2S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Zn
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 13 / Source method: obtained synthetically / Formula: C3H8O3
#5: Chemical...
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 22 / Source method: obtained synthetically / Formula: Cl
#6: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: SO4
#7: Chemical
ChemComp-KAI / 3-(CARBOXYMETHYL)-4-ISOPROPENYLPROLINE / KAINATE


Mass: 213.230 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H15NO4 / Feature type: SUBJECT OF INVESTIGATION / Comment: neurotransmitter, agonist*YM
#8: Chemical ChemComp-ACT / ACETATE ION


Mass: 59.044 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H3O2
#9: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 115 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.32 Å3/Da / Density % sol: 47.01 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: PEG 4000, lithium sulfate, zinc acetate, Tris buffer pH 8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: MAX II / Beamline: I911-3 / Wavelength: 1 Å
DetectorType: MARMOSAIC 225 mm CCD / Detector: CCD / Date: Mar 29, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.9→46.84 Å / Num. obs: 47190 / % possible obs: 99.9 % / Redundancy: 4.1 % / Rpim(I) all: 0.066 / Rrim(I) all: 0.133 / Net I/σ(I): 11.6 / Num. measured all: 192159
Reflection shellResolution: 2.9→3.06 Å / % possible obs: 99.7 % / Redundancy: 4.1 % / Num. measured all: 27910 / Num. unique obs: 6808 / Rpim(I) all: 0.245 / Rrim(I) all: 0.498 / Net I/σ(I) obs: 3.5

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Processing

Software
NameVersionClassification
PHENIX1.19.2_4158refinement
SCALAdata scaling
PHASERphasing
XDSdata reduction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.9→45.33 Å / SU ML: 0.37 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 23.99 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2345 2378 5.05 %
Rwork0.2068 --
obs0.2082 47131 99.77 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.9→45.33 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15885 0 420 115 16420
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00216665
X-RAY DIFFRACTIONf_angle_d0.49122485
X-RAY DIFFRACTIONf_dihedral_angle_d10.7626269
X-RAY DIFFRACTIONf_chiral_restr0.0412476
X-RAY DIFFRACTIONf_plane_restr0.0042799
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.9-2.960.34381430.29382610X-RAY DIFFRACTION100
2.96-3.020.31471410.27742602X-RAY DIFFRACTION100
3.02-3.090.32161330.27272600X-RAY DIFFRACTION100
3.09-3.170.27631370.25852627X-RAY DIFFRACTION100
3.17-3.260.26161300.24442628X-RAY DIFFRACTION100
3.26-3.350.26111440.24452615X-RAY DIFFRACTION100
3.35-3.460.261460.23972628X-RAY DIFFRACTION100
3.46-3.580.27361310.22772635X-RAY DIFFRACTION100
3.58-3.730.25041010.20872667X-RAY DIFFRACTION100
3.73-3.90.25211310.20172623X-RAY DIFFRACTION100
3.9-4.10.22381390.18462621X-RAY DIFFRACTION100
4.1-4.360.22441460.17572659X-RAY DIFFRACTION100
4.36-4.70.17141680.16142582X-RAY DIFFRACTION100
4.7-5.170.20061560.1592646X-RAY DIFFRACTION100
5.17-5.910.22431690.19772642X-RAY DIFFRACTION100
5.91-7.450.20821410.19452649X-RAY DIFFRACTION100
7.45-45.330.16911220.16982719X-RAY DIFFRACTION99
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)
12.212-0.1972-0.65111.93650.08822.70430.10210.17420.1004-0.42480.02370.35980.0774-0.40830.02890.3612-0.0162-0.09720.224-0.00310.2545-16.296623.6735-16.3907
22.58860.6909-0.24311.5576-0.21331.73140.02370.2026-0.10690.1873-0.0464-0.09680.11150.13310.00290.23220.0879-0.0350.1837-0.01630.2322-9.8756-26.5862-47.6533
31.3375-0.2327-0.55471.58520.64642.79750.11140.67820.2077-0.10080.08750.0488-0.2335-0.4510.03750.21680.0676-0.0040.49510.09340.2348-30.0036-14.0729-65.365
41.04620.0965-0.53871.8624-0.37343.11660.1289-0.110.2077-0.1210.1126-0.1653-0.24730.25960.03160.2371-0.03170.05050.2151-0.09870.2614.691534.25111.7185
53.2852-0.7751-0.34641.34040.82591.3040.0760.165-0.4133-0.088-0.03880.1150.1876-0.12770.00090.2359-0.0193-0.00070.17270.01580.2711-58.0109-30.4209-35.1696
61.41580.2164-0.3682.4243-1.0532.25850.0241-0.09280.03570.1003-0.0618-0.2122-0.13870.384-0.00540.29210.033-0.00060.2937-0.02910.267331.441417.323-30.2332
72.4625-0.271-0.60141.05830.36762.61530.00440.55610.1668-0.1109-0.13310.2606-0.0892-0.50650.00140.42650.066-0.01190.35990.02910.36239.251426.0923-47.7417
82.92090.502-0.62520.8087-0.2670.95310.0034-0.4464-0.27190.0618-0.0858-0.14620.08270.1801-0.01140.2130.0346-0.01010.24950.03620.3025-35.0515-25.5979-17.0089
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1(chain 'A' and resid 7 through 258)
2X-RAY DIFFRACTION2(chain 'B' and resid 7 through 253)
3X-RAY DIFFRACTION3(chain 'C' and resid 7 through 258)
4X-RAY DIFFRACTION4(chain 'D' and resid 6 through 258)
5X-RAY DIFFRACTION5(chain 'E' and resid 7 through 253)
6X-RAY DIFFRACTION6(chain 'F' and resid 7 through 254)
7X-RAY DIFFRACTION7(chain 'G' and resid 6 through 258)
8X-RAY DIFFRACTION8(chain 'H' and resid 6 through 253)

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