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- PDB-9eo4: Outward-open structure of human dopamine transporter bound to cocaine -

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Basic information

Entry
Database: PDB / ID: 9eo4
TitleOutward-open structure of human dopamine transporter bound to cocaine
ComponentsSodium-dependent dopamine transporter
KeywordsMEMBRANE PROTEIN / neurotransmitter/sodium symporters / monoamine transporter / dopamine transporter / SLC6
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 / adenohypophysis development / dopamine uptake / 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 / adenohypophysis development / dopamine uptake / hyaloid vascular plexus regression / dopamine binding / norepinephrine:sodium symporter activity / dopamine:sodium symporter activity / regulation of dopamine metabolic process / neurotransmitter transmembrane transporter activity / dopamine transport / flotillin complex / dopaminergic synapse / dopamine catabolic process / monoamine transmembrane transporter activity / monoamine transport / positive regulation of multicellular organism growth / Na+/Cl- dependent neurotransmitter transporters / response to iron ion / dopamine biosynthetic process / neurotransmitter transport / heterocyclic compound binding / dopamine uptake involved in synaptic transmission / amino acid transport / prepulse inhibition / response to cAMP / axon terminus / lactation / sodium ion transmembrane transport / protein phosphatase 2A binding / locomotory behavior / response to nicotine / response to cocaine / cognition / sensory perception of smell / presynaptic membrane / protease binding / response to ethanol / postsynaptic membrane / neuron projection / membrane raft / response to xenobiotic stimulus / axon / signaling receptor binding / 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
CHOLESTEROL / COCAINE / CHOLESTEROL HEMISUCCINATE / Sodium-dependent dopamine transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.66 Å
AuthorsNielsen, J.C. / Salomon, K. / Kalenderoglou, I.E. / Bargmeyer, S. / Pape, T. / Shahsavar, A. / Loland, C.J.
Funding support Denmark, European Union, 6items
OrganizationGrant numberCountry
LundbeckfondenR344-2020-1020 Denmark
The Carlsberg FoundationCF20-0345 Denmark
Independent Research Fund Denmark - Technology and Production Sciences3101-00381B Denmark
H2020 Marie Curie Actions of the European Commission860954European Union
Maersk FoundationL-2022-00324 Denmark
Maersk FoundationL-2021-00122 Denmark
CitationJournal: Nature / Year: 2024
Title: Structure of the human dopamine transporter in complex with cocaine.
Authors: Jeppe C Nielsen / Kristine Salomon / Iris E Kalenderoglou / Sarah Bargmeyer / Tillmann Pape / Azadeh Shahsavar / Claus J Loland /
Abstract: The dopamine transporter (DAT) is crucial for regulating dopamine signalling and is the prime mediator for the rewarding and addictive effects of cocaine. As part of the neurotransmitter sodium ...The dopamine transporter (DAT) is crucial for regulating dopamine signalling and is the prime mediator for the rewarding and addictive effects of cocaine. As part of the neurotransmitter sodium symporter family, DAT uses the Na gradient across cell membranes to transport dopamine against its chemical gradient. The transport mechanism involves both intra- and extracellular gates that control substrate access to a central site. However, the molecular intricacies of this process and the inhibitory mechanism of cocaine have remained unclear. Here, we present the molecular structure of human DAT in complex with cocaine at a resolution of 2.66 Å. Our findings reveal that DAT adopts the expected LeuT-fold, posing in an outward-open conformation with cocaine bound at the central (S1) site. Notably, while an Na occupies the second Na site (Na2), the Na1 site seems to be vacant, with the side chain of Asn82 occupying the presumed Na space. This structural insight elucidates the mechanism for the cocaine inhibition of human DAT and deepens our understanding of neurotransmitter transport. By shedding light on the molecular underpinnings of how cocaine acts, our study lays a foundation for the development of targeted medications to combat addiction.
History
DepositionMar 14, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 3, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2024Group: Data collection / Database references / Category: citation / em_admin
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Revision 1.2Aug 21, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._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 / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.4Nov 13, 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

Downloads & links

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Assembly

Deposited unit
B: Sodium-dependent dopamine transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)74,2188
Polymers72,2091
Non-polymers2,0087
Water724
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 , 1 types, 1 molecules B

#1: Protein Sodium-dependent dopamine transporter / DA transporter / DAT / Solute carrier family 6 member 3


Mass: 72209.281 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: triple tandem GGGS linker and Twin-Strep tag / Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A3, DAT1 / Production host: Homo sapiens (human) / References: UniProt: Q01959

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Non-polymers , 6 types, 11 molecules

#2: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H46O
#3: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#5: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#6: Chemical ChemComp-COC / COCAINE


Mass: 303.353 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C17H21NO4 / Feature type: SUBJECT OF INVESTIGATION / Comment: alkaloid*YM
#7: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 4 / 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: Human dopamine transporter bound to cocaine / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.72 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Calibrated magnification: 170700 X / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.27 sec. / Electron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 25808
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 5 eV

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPU3.6image acquisition
4cryoSPARCCTF correction
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1990718 / Symmetry type: POINT

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