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- PDB-7qhh: Desensitized state of GluA1/2 AMPA receptor in complex with TARP-... -

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Basic information

Entry
Database: PDB / ID: 7qhh
TitleDesensitized state of GluA1/2 AMPA receptor in complex with TARP-gamma 8 (TMD-LBD)
Components
  • (Isoform Flip of Glutamate receptor ...) x 2
  • Voltage-dependent calcium channel gamma-8 subunit
KeywordsMEMBRANE PROTEIN / glutamate / AMPA receptor / TARPs
Function / homology
Function and homology information


Phase 2 - plateau phase / Phase 0 - rapid depolarisation / Cargo concentration in the ER / cellular response to amine stimulus / axonal spine / COPII-mediated vesicle transport / positive regulation of membrane potential / chemical synaptic transmission, postsynaptic / cellular response to ammonium ion / neurotransmitter receptor transport, postsynaptic endosome to lysosome ...Phase 2 - plateau phase / Phase 0 - rapid depolarisation / Cargo concentration in the ER / cellular response to amine stimulus / axonal spine / COPII-mediated vesicle transport / positive regulation of membrane potential / chemical synaptic transmission, postsynaptic / cellular response to ammonium ion / neurotransmitter receptor transport, postsynaptic endosome to lysosome / L-type voltage-gated calcium channel complex / neurotransmitter receptor activity involved in regulation of postsynaptic cytosolic calcium ion concentration / LGI-ADAM interactions / neuron spine / myosin V binding / Trafficking of AMPA receptors / regulation of AMPA receptor activity / neurotransmitter receptor internalization / channel regulator activity / protein phosphatase 2B binding / dendritic spine membrane / postsynaptic neurotransmitter receptor diffusion trapping / response to arsenic-containing substance / cellular response to dsRNA / Synaptic adhesion-like molecules / long-term synaptic depression / beta-2 adrenergic receptor binding / protein kinase A binding / cellular response to peptide hormone stimulus / neuronal cell body membrane / spine synapse / spinal cord development / dendritic spine neck / dendritic spine head / Activation of AMPA receptors / response to lithium ion / perisynaptic space / cellular response to glycine / transmission of nerve impulse / AMPA glutamate receptor activity / regulation of postsynaptic membrane neurotransmitter receptor levels / Trafficking of GluR2-containing AMPA receptors / immunoglobulin binding / calcium channel regulator activity / AMPA glutamate receptor complex / neuronal action potential / kainate selective glutamate receptor activity / excitatory synapse / ionotropic glutamate receptor complex / extracellularly glutamate-gated ion channel activity / adenylate cyclase binding / cellular response to organic cyclic compound / asymmetric synapse / G-protein alpha-subunit binding / regulation of receptor recycling / Unblocking of NMDA receptors, glutamate binding and activation / long-term memory / voltage-gated calcium channel activity / glutamate receptor binding / regulation of postsynaptic membrane potential / positive regulation of synaptic transmission / response to electrical stimulus / 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 / synapse assembly / 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 / monoatomic ion transmembrane transport / positive regulation of synaptic transmission, glutamatergic / SNARE binding / dendritic shaft / response to cocaine / transmitter-gated monoatomic ion channel activity involved in regulation of postsynaptic membrane potential / synaptic membrane / synaptic transmission, glutamatergic / PDZ domain binding / cellular response to amino acid stimulus / postsynaptic density membrane / protein tetramerization / regulation of synaptic plasticity / modulation of chemical synaptic transmission / neuromuscular junction / Schaffer collateral - CA1 synapse / establishment of protein localization / terminal bouton / receptor internalization / synaptic vesicle membrane / response to organic cyclic compound / cerebral cortex development / response to peptide hormone / response to toxic substance / cellular response to growth factor stimulus
Similarity search - Function
Voltage-dependent calcium channel, gamma-8 subunit / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / 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 ...Voltage-dependent calcium channel, gamma-8 subunit / PMP-22/EMP/MP20/Claudin family / Voltage-dependent calcium channel, gamma subunit / PMP-22/EMP/MP20/Claudin superfamily / 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
(2S)-2,3-dihydroxypropyl (7Z)-hexadec-7-enoate / GLUTAMIC ACID / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / PALMITOLEIC ACID / Glutamate receptor 1 / Glutamate receptor 2 / Voltage-dependent calcium channel gamma-8 subunit
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsHerguedas, B. / Kohegyi, B. / Dohrke, J.N. / Watson, J.F. / Zhang, D. / Ho, H. / Shaikh, S. / Lape, R. / Krieger, J.M. / Greger, I.H.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_U105174197 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/N002113/1 United Kingdom
Spanish Ministry of Science, Innovation, and UniversitiesPID2019-106284GA-I00 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Mechanisms underlying TARP modulation of the GluA1/2-γ8 AMPA receptor.
Authors: Beatriz Herguedas / Bianka K Kohegyi / Jan-Niklas Dohrke / Jake F Watson / Danyang Zhang / Hinze Ho / Saher A Shaikh / Remigijus Lape / James M Krieger / Ingo H Greger /
Abstract: AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel ...AMPA-type glutamate receptors (AMPARs) mediate rapid signal transmission at excitatory synapses in the brain. Glutamate binding to the receptor's ligand-binding domains (LBDs) leads to ion channel activation and desensitization. Gating kinetics shape synaptic transmission and are strongly modulated by transmembrane AMPAR regulatory proteins (TARPs) through currently incompletely resolved mechanisms. Here, electron cryo-microscopy structures of the GluA1/2 TARP-γ8 complex, in both open and desensitized states (at 3.5 Å), reveal state-selective engagement of the LBDs by the large TARP-γ8 loop ('β1'), elucidating how this TARP stabilizes specific gating states. We further show how TARPs alter channel rectification, by interacting with the pore helix of the selectivity filter. Lastly, we reveal that the Q/R-editing site couples the channel constriction at the filter entrance to the gate, and forms the major cation binding site in the conduction path. Our results provide a mechanistic framework of how TARPs modulate AMPAR gating and conductance.
History
DepositionDec 12, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 23, 2022Provider: repository / Type: Initial release

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Isoform Flip of Glutamate receptor 1
B: Isoform Flip of Glutamate receptor 2
C: Isoform Flip of Glutamate receptor 1
D: Isoform Flip of Glutamate receptor 2
J: Voltage-dependent calcium channel gamma-8 subunit
I: Voltage-dependent calcium channel gamma-8 subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)489,47324
Polymers484,9706
Non-polymers4,50318
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22C
13A
23D
14B
24C
15B
25D
16C
26D
17J
27I

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A390 - 901
2010B394 - 901
1020A390 - 901
2020C390 - 901
1030A390 - 901
2030D394 - 901
1040B394 - 901
2040C390 - 901
1050B393 - 901
2050D393 - 901
1060C390 - 901
2060D394 - 901
1070J16 - 235
2070I16 - 235

NCS ensembles :
ID
1
2
3
4
5
6
7

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Components

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Isoform Flip of Glutamate receptor ... , 2 types, 4 molecules ACBD

#1: Protein Isoform Flip of Glutamate receptor 1 / GluR-1 / AMPA-selective glutamate receptor 1 / GluR-A / GluR-K1 / Glutamate receptor ionotropic / ...GluR-1 / AMPA-selective glutamate receptor 1 / GluR-A / GluR-K1 / Glutamate receptor ionotropic / AMPA 1 / GluA1


Mass: 102661.930 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria1, Glur1 / Cell line (production host): HEK293 Expi / Production host: Homo sapiens (human) / References: UniProt: P19490
#2: 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: 96247.055 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Gria2, Glur2 / Cell line (production host): HEK293 Expi / Production host: Homo sapiens (human) / References: UniProt: P19491

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Protein , 1 types, 2 molecules JI

#3: Protein Voltage-dependent calcium channel gamma-8 subunit / Neuronal voltage-gated calcium channel gamma-8 subunit / Transmembrane AMPAR regulatory protein ...Neuronal voltage-gated calcium channel gamma-8 subunit / Transmembrane AMPAR regulatory protein gamma-8 / TARP gamma-8


Mass: 43576.004 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Cacng8 / Cell line (production host): HEK293 Expi / Production host: Homo sapiens (human) / References: UniProt: Q8VHW5

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Non-polymers , 4 types, 18 molecules

#4: Chemical
ChemComp-GLU / GLUTAMIC ACID / Glutamic acid


Type: L-peptide linking / Mass: 147.129 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C5H9NO4 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-PAM / PALMITOLEIC ACID / Palmitoleic acid


Mass: 254.408 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C16H30O2
#6: Chemical ChemComp-OLC / (2R)-2,3-dihydroxypropyl (9Z)-octadec-9-enoate / 1-Oleoyl-R-glycerol


Mass: 356.540 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C21H40O4
#7: Chemical ChemComp-79N / (2S)-2,3-dihydroxypropyl (7Z)-hexadec-7-enoate


Mass: 328.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C19H36O4

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Details

Has ligand of interestY

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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: Complex between GluA1/2 AMPA receptor and auxiliary subunit TARP gamma8
Type: COMPLEX
Details: GluA2 and TARP8 are expressed as a tandem construct
Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.490 MDa / Experimental value: NO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 EXPI / Plasmid: PRK5
Buffer solutionpH: 8
Details: 25 mM TRIS 150 mM NaCl 0.02 % GDN 87 uM CTZ 100 mM L-Glu
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was incubated with 87 uM CTZ for 30 minutes and 100 mM L-Glutamate was added before grid preparation
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 3-4 second blots

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 340 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 51 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9664

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4GctfCTF correctionGctf was used to performed CTF correction
5RELION3.1CTF correctionCTF refinement was performed with relion
8Cootmodel fitting
9UCSF Chimeramodel fitting
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
15REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 105918 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: First, rigid body fit was performed in Chimera using the coordinates of the TMD of 6QKC and the coordinates of the LBD in PDB code 3TKD. Manual model building was performed with Coot and ...Details: First, rigid body fit was performed in Chimera using the coordinates of the TMD of 6QKC and the coordinates of the LBD in PDB code 3TKD. Manual model building was performed with Coot and Refinement was performed with Refmac. The TMD and LBD maps obtained independently aided model building but were not used for refinement.
Atomic model building
IDPDB-ID 3D fitting-ID
16QKC

6qkc

1
23TKD

3tkd

1
RefinementResolution: 3.6→339.2 Å / Cor.coef. Fo:Fc: 0.664 / SU B: 23.138 / SU ML: 0.306 / ESU R: 0.153
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.47551 --
obs0.47551 1752135 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 81.995 Å2
Baniso -1Baniso -2Baniso -3
1--0.1 Å2-0.99 Å20 Å2
2---1.09 Å2-0 Å2
3---1.2 Å2
Refinement stepCycle: 1 / Total: 13622
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0060.01313902
ELECTRON MICROSCOPYr_bond_other_d00.01712100
ELECTRON MICROSCOPYr_angle_refined_deg1.2631.62818978
ELECTRON MICROSCOPYr_angle_other_deg1.3811.56427348
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.86351960
ELECTRON MICROSCOPYr_dihedral_angle_2_deg24.54120.433416
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.195151462
ELECTRON MICROSCOPYr_dihedral_angle_4_deg7.5841538
ELECTRON MICROSCOPYr_chiral_restr0.0550.21990
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0216362
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023226
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it2.80211.4227906
ELECTRON MICROSCOPYr_mcbond_other2.80211.4217905
ELECTRON MICROSCOPYr_mcangle_it5.31217.1159844
ELECTRON MICROSCOPYr_mcangle_other5.31117.1179845
ELECTRON MICROSCOPYr_scbond_it1.3939.0075996
ELECTRON MICROSCOPYr_scbond_other1.3939.0065997
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other3.1314.1929135
ELECTRON MICROSCOPYr_long_range_B_refined16.28554678
ELECTRON MICROSCOPYr_long_range_B_other16.28554679
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A180720.16
12B180720.16
21A215660
22C215660
31A180740.16
32D180740.16
41B180720.16
42C180720.16
51B216520
52D216520
61C180740.16
62D180740.16
71J109440
72I109440
LS refinement shellResolution: 3.6→3.694 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.553 130198 -
obs--100 %

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