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Yorodumi- PDB-7sk2: Human wildtype GABA reuptake transporter 1 in complex with tiagab... -
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-Basic information
Entry | Database: PDB / ID: 7sk2 | ||||||
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Title | Human wildtype GABA reuptake transporter 1 in complex with tiagabine, inward-open conformation | ||||||
Components | Sodium- and chloride-dependent GABA transporter 1 | ||||||
Keywords | MEMBRANE PROTEIN / Neurotransmitter transporter | ||||||
Function / homology | Function and homology information gamma-aminobutyric acid reuptake / Reuptake of GABA / gamma-aminobutyric acid:sodium:chloride symporter activity / sodium:chloride symporter activity / gamma-aminobutyric acid transmembrane transporter activity / gamma-aminobutyric acid import / inorganic anion import across plasma membrane / negative regulation of synaptic transmission, GABAergic / positive regulation of gamma-aminobutyric acid secretion / response to purine-containing compound ...gamma-aminobutyric acid reuptake / Reuptake of GABA / gamma-aminobutyric acid:sodium:chloride symporter activity / sodium:chloride symporter activity / gamma-aminobutyric acid transmembrane transporter activity / gamma-aminobutyric acid import / inorganic anion import across plasma membrane / negative regulation of synaptic transmission, GABAergic / positive regulation of gamma-aminobutyric acid secretion / response to purine-containing compound / response to sucrose / sodium ion import across plasma membrane / Na+/Cl- dependent neurotransmitter transporters / amino acid transport / associative learning / transport across blood-brain barrier / sodium ion transmembrane transport / GABA-ergic synapse / chloride transmembrane transport / response to cocaine / response to lead ion / synapse organization / memory / response to toxic substance / response to calcium ion / response to estradiol / presynaptic membrane / chemical synaptic transmission / postsynaptic membrane / axon / neuronal cell body / cell surface / identical protein binding / membrane / metal ion binding / plasma membrane Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.82 Å | ||||||
Authors | Gati, C. / Motiwala, Z. / Aduri, N.G. / Shaye, H. / Han, G.W. / Cherezov, V. | ||||||
Funding support | 1items
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Citation | Journal: Nature / Year: 2022 Title: Structural basis of GABA reuptake inhibition. Authors: Zenia Motiwala / Nanda Gowtham Aduri / Hamidreza Shaye / Gye Won Han / Jordy Homing Lam / Vsevolod Katritch / Vadim Cherezov / Cornelius Gati / Abstract: γ-Aminobutyric acid (GABA) transporter 1 (GAT1) regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release ...γ-Aminobutyric acid (GABA) transporter 1 (GAT1) regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7sk2.cif.gz | 101.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb7sk2.ent.gz | 78.2 KB | Display | PDB format |
PDBx/mmJSON format | 7sk2.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 7sk2_validation.pdf.gz | 843.1 KB | Display | wwPDB validaton report |
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Full document | 7sk2_full_validation.pdf.gz | 847.5 KB | Display | |
Data in XML | 7sk2_validation.xml.gz | 22.4 KB | Display | |
Data in CIF | 7sk2_validation.cif.gz | 31.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/sk/7sk2 ftp://data.pdbj.org/pub/pdb/validation_reports/sk/7sk2 | HTTPS FTP |
-Related structure data
Related structure data | 25170MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 64886.793 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A1, GABATR, GABT1, GAT1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P30531 |
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#2: Chemical | ChemComp-TGI / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: GAT-1 in complex with tiagabine / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 67 kDa/nm / Experimental value: YES | ||||||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | ||||||||||||||||||||
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 18 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 298 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 105000 X / Nominal defocus max: 25000 nm / Nominal defocus min: 10000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 39572 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
Software | Name: PHENIX / Version: 1.18_3861: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 135297 / Algorithm: FOURIER SPACE / Symmetry type: POINT | ||||||||||||||||||||||||
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