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- PDB-6uo8: Human metabotropic GABA(B) receptor bound to agonist SKF97541 and... -

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Basic information

Entry
Database: PDB / ID: 6uo8
TitleHuman metabotropic GABA(B) receptor bound to agonist SKF97541 and positive allosteric modulator GS39783
Components(Gamma-aminobutyric acid type B receptor subunit ...) x 2
KeywordsMEMBRANE PROTEIN / G protein-coupled receptor / GABA / GABAB / PAM / Neurotransmitter
Function / homology
Function and homology information


G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / GABA B receptor activation / G protein-coupled neurotransmitter receptor activity involved in regulation of presynaptic membrane potential / G protein-coupled GABA receptor complex / negative regulation of gamma-aminobutyric acid secretion / neuron-glial cell signaling / G protein-coupled GABA receptor activity / G protein-coupled receptor heterodimeric complex / negative regulation of epinephrine secretion / negative regulation of dopamine secretion ...G protein-coupled neurotransmitter receptor activity involved in regulation of postsynaptic membrane potential / GABA B receptor activation / G protein-coupled neurotransmitter receptor activity involved in regulation of presynaptic membrane potential / G protein-coupled GABA receptor complex / negative regulation of gamma-aminobutyric acid secretion / neuron-glial cell signaling / G protein-coupled GABA receptor activity / G protein-coupled receptor heterodimeric complex / negative regulation of epinephrine secretion / negative regulation of dopamine secretion / positive regulation of growth hormone secretion / extracellular matrix protein binding / GABA receptor complex / negative regulation of adenylate cyclase activity / Class C/3 (Metabotropic glutamate/pheromone receptors) / gamma-aminobutyric acid signaling pathway / synaptic transmission, GABAergic / positive regulation of glutamate secretion / negative regulation of synaptic transmission / axolemma / GABA-ergic synapse / adenylate cyclase-inhibiting G protein-coupled receptor signaling pathway / dendritic shaft / response to nicotine / mitochondrial membrane / Schaffer collateral - CA1 synapse / Activation of G protein gated Potassium channels / Inhibition of voltage gated Ca2+ channels via Gbeta/gamma subunits / osteoblast differentiation / transmembrane signaling receptor activity / synaptic vesicle / presynaptic membrane / G alpha (i) signalling events / chemical synaptic transmission / postsynaptic membrane / response to ethanol / dendritic spine / neuron projection / G protein-coupled receptor signaling pathway / protein heterodimerization activity / negative regulation of cell population proliferation / neuronal cell body / glutamatergic synapse / endoplasmic reticulum membrane / extracellular space / plasma membrane / cytoplasm
Similarity search - Function
GPCR family 3, gamma-aminobutyric acid receptor, type B2 / Gamma-aminobutyric acid type B receptor subunit 2, coiled-coil domain / Gamma-aminobutyric acid type B receptor subunit 2 coiled-coil domain / GPCR family 3, gamma-aminobutyric acid receptor, type B1 / GPCR family 3, GABA-B receptor / GPCR, family 3, conserved site / G-protein coupled receptors family 3 signature 3. / GPCR, family 3 / GPCR family 3, C-terminal / 7 transmembrane sweet-taste receptor of 3 GCPR ...GPCR family 3, gamma-aminobutyric acid receptor, type B2 / Gamma-aminobutyric acid type B receptor subunit 2, coiled-coil domain / Gamma-aminobutyric acid type B receptor subunit 2 coiled-coil domain / GPCR family 3, gamma-aminobutyric acid receptor, type B1 / GPCR family 3, GABA-B receptor / GPCR, family 3, conserved site / G-protein coupled receptors family 3 signature 3. / GPCR, family 3 / GPCR family 3, C-terminal / 7 transmembrane sweet-taste receptor of 3 GCPR / G-protein coupled receptors family 3 profile. / Sushi repeat (SCR repeat) / Domain abundant in complement control proteins; SUSHI repeat; short complement-like repeat (SCR) / Sushi/SCR/CCP domain / Sushi/SCR/CCP superfamily / Sushi/CCP/SCR domain profile. / Receptor, ligand binding region / Receptor family ligand binding region / Periplasmic binding protein-like I
Similarity search - Domain/homology
(R)-(3-aminopropyl)methylphosphinic acid / Chem-QDA / Gamma-aminobutyric acid type B receptor subunit 2 / Gamma-aminobutyric acid type B receptor subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.63 Å
AuthorsShaye, H. / Han, G.W. / Gati, C. / Cherezov, V.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127086 United States
Department of Energy (DOE, United States)DE-AC02-76SF00515 United States
CitationJournal: Nature / Year: 2020
Title: Structural basis of the activation of a metabotropic GABA receptor.
Authors: Hamidreza Shaye / Andrii Ishchenko / Jordy Homing Lam / Gye Won Han / Li Xue / Philippe Rondard / Jean-Philippe Pin / Vsevolod Katritch / Cornelius Gati / Vadim Cherezov /
Abstract: Metabotropic γ-aminobutyric acid receptors (GABA) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that ...Metabotropic γ-aminobutyric acid receptors (GABA) are involved in the modulation of synaptic responses in the central nervous system and have been implicated in neuropsychological conditions that range from addiction to psychosis. GABA belongs to class C of the G-protein-coupled receptors, and its functional entity comprises an obligate heterodimer that is composed of the GB1 and GB2 subunits. Each subunit possesses an extracellular Venus flytrap domain, which is connected to a canonical seven-transmembrane domain. Here we present four cryo-electron microscopy structures of the human full-length GB1-GB2 heterodimer: one structure of its inactive apo state, two intermediate agonist-bound forms and an active form in which the heterodimer is bound to an agonist and a positive allosteric modulator. The structures reveal substantial differences, which shed light on the complex motions that underlie the unique activation mechanism of GABA. Our results show that agonist binding leads to the closure of the Venus flytrap domain of GB1, triggering a series of transitions, first rearranging and bringing the two transmembrane domains into close contact along transmembrane helix 6 and ultimately inducing conformational rearrangements in the GB2 transmembrane domain via a lever-like mechanism to initiate downstream signalling. This active state is stabilized by a positive allosteric modulator binding at the transmembrane dimerization interface.
History
DepositionOct 14, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 1, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / em_entity_assembly / 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.type_symbol / _chem_comp.name / _em_entity_assembly.entity_id_list / _pdbx_struct_assembly_gen.asym_id_list / _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 2.1Aug 26, 2020Group: Database references / Structure summary / Category: chem_comp / citation
Item: _chem_comp.pdbx_synonyms / _citation.journal_volume ..._chem_comp.pdbx_synonyms / _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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Assembly

Deposited unit
A: Gamma-aminobutyric acid type B receptor subunit 1
B: Gamma-aminobutyric acid type B receptor subunit 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)177,88913
Polymers174,6522
Non-polymers3,23711
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Only dimeric fractions were pooled after size exclusion chromatography. Moreover, only dimeric species were selected for 3D reconstruction.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5890 Å2
ΔGint8 kcal/mol
Surface area72520 Å2

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Components

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Gamma-aminobutyric acid type B receptor subunit ... , 2 types, 2 molecules AB

#1: Protein Gamma-aminobutyric acid type B receptor subunit 1 / Gb1


Mass: 86450.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABBR1, GPRC3A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9UBS5
#2: Protein Gamma-aminobutyric acid type B receptor subunit 2 / Gb2 / G-protein coupled receptor 51 / HG20


Mass: 88200.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GABBR2, GPR51, GPRC3B / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O75899

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Sugars , 3 types, 9 molecules

#3: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2 / Source method: obtained synthetically
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#4: 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.: 1 / Source method: obtained synthetically
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
#6: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-QD7 / (R)-(3-aminopropyl)methylphosphinic acid / SKF-97,541


Mass: 137.117 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO2P / Feature type: SUBJECT OF INVESTIGATION / Comment: agonist*YM
#7: Chemical ChemComp-QDA / N~4~,N~6~-dicyclopentyl-2-(methylsulfanyl)-5-nitropyrimidine-4,6-diamine / GS-39783


Mass: 337.440 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H23N5O2S / Feature type: SUBJECT OF INVESTIGATION

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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: GABA(B) receptor bound to agonist and PAM / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.174 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human) / Cellular location: Membrane
Source (recombinant)Organism: Spodoptera aff. frugiperda 1 BOLD-2017 (butterflies/moths)
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 293.15 K

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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: 29000 X / Calibrated magnification: 29000 X / Nominal defocus max: -3000 nm / Nominal defocus min: -1500 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 recordingAverage exposure time: 0.07 sec. / Electron dose: 1.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10917

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
1cryoSPARC2.12.14particle selection
2SerialEM3.6image acquisition
4cryoSPARC2.12.14CTF correction
7PHENIX1.17.1-3660model fitting
9PHENIX1.17.1-3660model refinement
10cryoSPARC2.12.14initial Euler assignment
11cryoSPARC2.12.14final Euler assignment
12cryoSPARC2.12.14classification
13cryoSPARC2.12.143D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4173286
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89001 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
14MS3

4ms3

1
24OR2

4or2

1
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 116.49 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003611212
ELECTRON MICROSCOPYf_angle_d0.71315234
ELECTRON MICROSCOPYf_chiral_restr0.05061760
ELECTRON MICROSCOPYf_plane_restr0.00581914
ELECTRON MICROSCOPYf_dihedral_angle_d17.094022

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