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- PDB-9eup: Inhibitor-free outward-open structure of Drosophila dopamine tran... -

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Basic information

Entry
Database: PDB / ID: 9eup
TitleInhibitor-free outward-open structure of Drosophila dopamine transporter
Components
  • (9D5 ANTIBODY, ...) x 2
  • Sodium-dependent dopamine transporter
KeywordsMEMBRANE PROTEIN / SLC6A3 / Dopamine transporter / neurotransmitter sodium symporters
Function / homology
Function and homology information


Dopamine clearance from the synaptic cleft / Na+/Cl- dependent neurotransmitter transporters / circadian sleep/wake cycle / cocaine binding / dopamine:sodium symporter activity / norepinephrine:sodium symporter activity / norepinephrine transport / response to odorant / regulation of presynaptic cytosolic calcium ion concentration / dopamine transport ...Dopamine clearance from the synaptic cleft / Na+/Cl- dependent neurotransmitter transporters / circadian sleep/wake cycle / cocaine binding / dopamine:sodium symporter activity / norepinephrine:sodium symporter activity / norepinephrine transport / response to odorant / regulation of presynaptic cytosolic calcium ion concentration / dopamine transport / sleep / dopamine uptake involved in synaptic transmission / amino acid transport / neuronal cell body membrane / sodium ion transmembrane transport / adult locomotory behavior / presynaptic membrane / axon / metal ion binding / plasma membrane
Similarity search - Function
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
TRIS-HYDROXYMETHYL-METHYL-AMMONIUM / CHOLESTEROL / CHOLESTEROL HEMISUCCINATE / Sodium-dependent dopamine transporter
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsPedersen, C.N. / Yang, F. / Ita, S. / Xu, Y. / Akunuri, R. / Trampari, S. / Neumann, C.M.T. / Desdorf, L.M. / Schioett, B. / Salvino, J.M. ...Pedersen, C.N. / Yang, F. / Ita, S. / Xu, Y. / Akunuri, R. / Trampari, S. / Neumann, C.M.T. / Desdorf, L.M. / Schioett, B. / Salvino, J.M. / Mortensen, O.V. / Nissen, P. / Shahsavar, A.
Funding support Denmark, United States, 11items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF19OC0054875 Denmark
LundbeckfondenR310-2018-3713 Denmark
Danish Ministry for Research and Higher Education5072-00025B Denmark
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH121453 United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)MH106912 United States
LundbeckfondenR368-2021-522 Denmark
Novo Nordisk FoundationNNF18OC0032608 Denmark
Aarhus University Research FoundationAUFF-E-2022-9-24 Denmark
National Institutes of Health/National Library of Medicine (NIH/NLM)R01 DA051205-03 United States
National Institutes of Health/National Library of Medicine (NIH/NLM)S10OD030245-01 United States
National Institutes of Health/National Library of Medicine (NIH/NLM)P30 CA010815-53 United States
CitationJournal: J Neurochem / Year: 2024
Title: Cryo-EM structure of the dopamine transporter with a novel atypical non-competitive inhibitor bound to the orthosteric site.
Authors: Clara Nautrup Pedersen / Fuyu Yang / Samantha Ita / Yibin Xu / Ravikumar Akunuri / Sofia Trampari / Caroline Marie Teresa Neumann / Lasse Messell Desdorf / Birgit Schiøtt / Joseph M Salvino ...Authors: Clara Nautrup Pedersen / Fuyu Yang / Samantha Ita / Yibin Xu / Ravikumar Akunuri / Sofia Trampari / Caroline Marie Teresa Neumann / Lasse Messell Desdorf / Birgit Schiøtt / Joseph M Salvino / Ole Valente Mortensen / Poul Nissen / Azadeh Shahsavar /
Abstract: The regulation of dopamine (DA) removal from the synaptic cleft is a crucial process in neurotransmission and is facilitated by the sodium- and chloride-coupled dopamine transporter DAT. ...The regulation of dopamine (DA) removal from the synaptic cleft is a crucial process in neurotransmission and is facilitated by the sodium- and chloride-coupled dopamine transporter DAT. Psychostimulant drugs, cocaine, and amphetamine, both block the uptake of DA, while amphetamine also triggers the release of DA. As a result, they prolong or even amplify neurotransmitter signaling. Atypical inhibitors of DAT lack cocaine-like rewarding effects and offer a promising strategy for the treatment of drug use disorders. Here, we present the 3.2 Å resolution cryo-electron microscopy structure of the Drosophila melanogaster dopamine transporter (dDAT) in complex with the atypical non-competitive inhibitor AC-4-248. The inhibitor partially binds at the central binding site, extending into the extracellular vestibule, and locks the transporter in an outward open conformation. Our findings propose mechanisms for the non-competitive inhibition of DAT and attenuation of cocaine potency by AC-4-248 and provide a basis for the rational design of more efficacious atypical inhibitors.
History
DepositionMar 27, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium-dependent dopamine transporter
H: 9D5 ANTIBODY, HEAVY CHAIN
L: 9D5 ANTIBODY, LIGHT CHAIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)113,7789
Polymers112,7013
Non-polymers1,0776
Water00
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 A

#1: Protein Sodium-dependent dopamine transporter / Protein fumin


Mass: 60939.520 Da / Num. of mol.: 1 / Mutation: V74A, V275A, V311A, L415A, G538L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Gene: DAT, fmn, CG8380 / Production host: Homo sapiens (human) / References: UniProt: Q7K4Y6

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Antibody , 2 types, 2 molecules HL

#2: Antibody 9D5 ANTIBODY, HEAVY CHAIN


Mass: 25921.338 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)
#3: Antibody 9D5 ANTIBODY, LIGHT CHAIN


Mass: 25840.607 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)

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Non-polymers , 5 types, 6 molecules

#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C31H50O4
#5: Chemical ChemComp-144 / TRIS-HYDROXYMETHYL-METHYL-AMMONIUM


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3
#6: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O
#7: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Na
#8: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl

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Details

Has ligand of interestN

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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: Inhibitor-free Drosophila melanogaster dopamine transporter in complex with Fab 9D5
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.107602 MDa / Experimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMsodium chlorideNaCl1
220 mMTris hydrochlorideTris-HCl1
30.01 (w/v) %Lauryl Maltose Neopentyl GlycolLMNG1
40.001 (w/v) %cholesterol-hemisuccinateCHS1
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3
VitrificationCryogen 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 defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4294

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
7Cootmodel fitting
8UCSF ChimeraXmodel fitting
13cryoSPARC3D reconstruction
14PHENIX1.20.1-4487model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 137600 / Symmetry type: POINT
Atomic model buildingPDB-ID: 4M48

4m48


Accession code: 4M48 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0087833
ELECTRON MICROSCOPYf_angle_d0.84410687
ELECTRON MICROSCOPYf_dihedral_angle_d8.0241083
ELECTRON MICROSCOPYf_chiral_restr0.0491184
ELECTRON MICROSCOPYf_plane_restr0.0091327

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