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- PDB-8vby: Structure of the human dopamine transporter in complex with beta-... -

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Basic information

Entry
Database: PDB / ID: 8vby
TitleStructure of the human dopamine transporter in complex with beta-CFT, MRS7292 and divalent zinc
ComponentsSodium-dependent dopamine transporter
KeywordsMEMBRANE PROTEIN / Transport / Membrane / Inhibitor
Function / homology
Function and homology information


Defective SLC6A3 causes Parkinsonism-dystonia infantile (PKDYS) / Defective SLC6A3 causes Parkinsonism-dystonia infantile (PKDYS) / Dopamine clearance from the synaptic cleft / amine binding / dopamine uptake / adenohypophysis development / hyaloid vascular plexus regression / dopamine binding / norepinephrine:sodium symporter activity / dopamine:sodium symporter activity ...Defective SLC6A3 causes Parkinsonism-dystonia infantile (PKDYS) / Defective SLC6A3 causes Parkinsonism-dystonia infantile (PKDYS) / Dopamine clearance from the synaptic cleft / amine binding / dopamine uptake / adenohypophysis development / hyaloid vascular plexus regression / dopamine binding / norepinephrine:sodium symporter activity / dopamine:sodium symporter activity / neurotransmitter transmembrane transporter activity / regulation of dopamine metabolic process / dopamine transport / flotillin complex / dopamine catabolic process / dopaminergic synapse / monoamine transmembrane transporter activity / monoamine transport / positive regulation of multicellular organism growth / SLC-mediated transport of neurotransmitters / heterocyclic compound binding / response to iron ion / dopamine biosynthetic process / neurotransmitter transport / amino acid transport / dopamine uptake involved in synaptic transmission / response to cAMP / prepulse inhibition / lactation / axon terminus / sodium ion transmembrane transport / protein phosphatase 2A binding / response to nicotine / response to cocaine / locomotory behavior / cognition / sensory perception of smell / presynaptic membrane / protease binding / response to ethanol / postsynaptic membrane / neuron projection / membrane raft / response to xenobiotic stimulus / signaling receptor binding / axon / neuronal cell body / protein-containing complex binding / cell surface / metal ion binding / membrane / plasma membrane / cytoplasm
Similarity search - Function
Sodium:neurotransmitter symporter, dopamine / Sodium:neurotransmitter symporter family signature 2. / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family / Sodium:neurotransmitter symporter family profile.
Similarity search - Domain/homology
Chem-42L / : / DECANE / DODECANE / HEPTANE / PENTANE / PENTADECANE / N-OCTANE / CHOLESTEROL HEMISUCCINATE / Sodium-dependent dopamine transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsSrivastava, D.K. / Gouaux, E.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)5R01MH070039 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Nature / Year: 2024
Title: Structure of the human dopamine transporter and mechanisms of inhibition.
Authors: Dushyant Kumar Srivastava / Vikas Navratna / Dilip K Tosh / Audrey Chinn / Md Fulbabu Sk / Emad Tajkhorshid / Kenneth A Jacobson / Eric Gouaux /
Abstract: The neurotransmitter dopamine has central roles in mood, appetite, arousal and movement. Despite its importance in brain physiology and function, and as a target for illicit and therapeutic drugs, ...The neurotransmitter dopamine has central roles in mood, appetite, arousal and movement. Despite its importance in brain physiology and function, and as a target for illicit and therapeutic drugs, the human dopamine transporter (hDAT) and mechanisms by which it is inhibited by small molecules and Zn are without a high-resolution structural context. Here we determine the structure of hDAT in a tripartite complex with the competitive inhibitor and cocaine analogue, (-)-2-β-carbomethoxy-3-β-(4-fluorophenyl)tropane (β-CFT), the non-competitive inhibitor MRS7292 and Zn (ref. ). We show how β-CFT occupies the central site, approximately halfway across the membrane, stabilizing the transporter in an outward-open conformation. MRS7292 binds to a structurally uncharacterized allosteric site, adjacent to the extracellular vestibule, sequestered underneath the extracellular loop 4 (EL4) and adjacent to transmembrane helix 1b (TM1b), acting as a wedge, precluding movement of TM1b and closure of the extracellular gate. A Zn ion further stabilizes the outward-facing conformation by coupling EL4 to EL2, TM7 and TM8, thus providing specific insights into how Zn restrains the movement of EL4 relative to EL2 and inhibits transport activity.
History
DepositionDec 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 7, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 14, 2024Group: Data collection / Structure summary / Category: em_admin / struct
Item: _em_admin.last_update / _em_admin.title / _struct.title
Revision 1.2Aug 21, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.3Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.4Sep 4, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.5Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium-dependent dopamine transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)70,28642
Polymers63,3851
Non-polymers6,90241
Water18010
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Sodium-dependent dopamine transporter


Mass: 63384.500 Da / Num. of mol.: 1 / Mutation: I248Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A3 / Production host: Homo sapiens (human) / References: UniProt: Q01959
#4: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / 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 , 12 types, 50 molecules

#2: Chemical ChemComp-42L / methyl (1R,2S,3S,5S)-3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate


Mass: 277.334 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H20FNO2 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-A1AAF / methyl (1S,2R,3S,4R,5R)-4-{2-[(5-chlorothiophen-2-yl)ethynyl]-6-(methylamino)-9H-purin-9-yl}-2,3-dihydroxybicyclo[3.1.0]hexane-1-carboxylate


Mass: 459.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H18ClN5O4S / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C31H50O4
#6: Chemical
ChemComp-D10 / DECANE


Mass: 142.282 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H22
#7: Chemical
ChemComp-LNK / PENTANE


Mass: 72.149 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C5H12
#8: Chemical
ChemComp-HP6 / HEPTANE


Mass: 100.202 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C7H16
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#10: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#11: Chemical
ChemComp-D12 / DODECANE


Mass: 170.335 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C12H26
#12: Chemical ChemComp-MYS / PENTADECANE


Mass: 212.415 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H32
#13: Chemical ChemComp-OCT / N-OCTANE


Mass: 114.229 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H18
#14: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 10 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: Sodium dependent dopamine transporter in complex with inhibitor
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.063 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMHEPESHEPES1
2200 mMSodium ChlorideNaCl1
30.02 %DetergentDigitonin1
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse sample
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 6 eV

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Processing

EM softwareName: cryoSPARC / Category: CTF correction
Image processingDetails: Falcon 4i
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 177494 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 53.23 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00264974
ELECTRON MICROSCOPYf_angle_d0.45836697
ELECTRON MICROSCOPYf_chiral_restr0.0364738
ELECTRON MICROSCOPYf_plane_restr0.004771
ELECTRON MICROSCOPYf_dihedral_angle_d6.8342836

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