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- EMDB-19978: Outward-open structure of Drosophila dopamine transporter bound t... -

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Basic information

Entry
Database: EMDB / ID: EMD-19978
TitleOutward-open structure of Drosophila dopamine transporter bound to an atypical non-competitive inhibitor
Map data
Sample
  • Complex: Drosophila melanogaster dopamine transporter bound to atypical inhibitor AC-4-248 and in complex with Fab 9D5
    • Protein or peptide: Sodium-dependent dopamine transporter
    • Protein or peptide: 9D5 ANTIBODY, LIGHT CHAIN
    • Protein or peptide: 9D5 ANTIBODY, HEAVY CHAIN
  • Ligand: N-[3-(6-chloranyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-2-yl)propyl]butanamide
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: TRIS-HYDROXYMETHYL-METHYL-AMMONIUM
  • Ligand: CHOLESTEROL
  • Ligand: SODIUM ION
  • Ligand: CHLORIDE ION
KeywordsSLC6A3 / Dopamine transporter / atypical inhibitors / neurotransmitter sodium symporters / MEMBRANE PROTEIN
Function / homology
Function and homology information


Dopamine clearance from the synaptic cleft / Na+/Cl- dependent neurotransmitter transporters / circadian sleep/wake cycle / cocaine binding / response to odorant / regulation of presynaptic cytosolic calcium ion concentration / dopamine:sodium symporter activity / norepinephrine transport / dopamine transport / sleep ...Dopamine clearance from the synaptic cleft / Na+/Cl- dependent neurotransmitter transporters / circadian sleep/wake cycle / cocaine binding / response to odorant / regulation of presynaptic cytosolic calcium ion concentration / dopamine:sodium symporter activity / norepinephrine transport / dopamine transport / sleep / amino acid transport / dopamine uptake involved in synaptic transmission / 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
Sodium-dependent dopamine transporter
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPedersen CN / Yang F / Ita S / Xu Y / Akunuri R / Trampari S / Neumann CMT / Desdorf LM / Schioett B / Salvino JM ...Pedersen CN / Yang F / Ita S / Xu Y / Akunuri R / Trampari S / Neumann CMT / Desdorf LM / Schioett B / Salvino JM / Mortensen OV / Nissen P / Shahsavar A
Funding support Denmark, United States, 11 items
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, 2024-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19978.png

Downloads & links

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Map

FileDownload / File: emd_19978.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.89 Å/pix.
x 256 pix.
= 227.744 Å		0.89 Å/pix.
x 256 pix.
= 227.744 Å		0.89 Å/pix.
x 256 pix.
= 227.744 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.88963 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.7
Minimum - Maximum-27.135525000000001 - 39.53584
Average (Standard dev.)-0.000000000001047 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 227.744 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map A

Fileemd_19978_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_19978_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Drosophila melanogaster dopamine transporter bound to atypical in...

EntireName: Drosophila melanogaster dopamine transporter bound to atypical inhibitor AC-4-248 and in complex with Fab 9D5
Components
  • Complex: Drosophila melanogaster dopamine transporter bound to atypical inhibitor AC-4-248 and in complex with Fab 9D5
    • Protein or peptide: Sodium-dependent dopamine transporter
    • Protein or peptide: 9D5 ANTIBODY, LIGHT CHAIN
    • Protein or peptide: 9D5 ANTIBODY, HEAVY CHAIN
  • Ligand: N-[3-(6-chloranyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-2-yl)propyl]butanamide
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: TRIS-HYDROXYMETHYL-METHYL-AMMONIUM
  • Ligand: CHOLESTEROL
  • Ligand: SODIUM ION
  • Ligand: CHLORIDE ION

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Supramolecule #1: Drosophila melanogaster dopamine transporter bound to atypical in...

SupramoleculeName: Drosophila melanogaster dopamine transporter bound to atypical inhibitor AC-4-248 and in complex with Fab 9D5
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 85.622 KDa

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Macromolecule #1: Sodium-dependent dopamine transporter

MacromoleculeName: Sodium-dependent dopamine transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 60.93952 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MNSISDERET WSGKVDFLLS VIGFAVDLAN VWRFPYLCYK NGGGAFLVPY GIMLAVGGIP LFYMELALGQ HNRKGAITCW GRLVPLFKG IGYAVVLIAF YVDFYYNVII AWSLRFFFAS FTNSLPWTSC NNIWNTPNCR PFESQGFQSA ASEYFNRYIL E LNRSEGIH ...String:
MNSISDERET WSGKVDFLLS VIGFAVDLAN VWRFPYLCYK NGGGAFLVPY GIMLAVGGIP LFYMELALGQ HNRKGAITCW GRLVPLFKG IGYAVVLIAF YVDFYYNVII AWSLRFFFAS FTNSLPWTSC NNIWNTPNCR PFESQGFQSA ASEYFNRYIL E LNRSEGIH DLGAIKWDMA LCLLIVYLIC YFSLWKGIST SGKVVWFTAL FPYAALLILL IRGLTLPGSF LGIQYYLTPN FS AIYKAEV WADAATQVFF SLGPGFGVLL AYASYNKYHN NVYKDALLTS FINSATSFIA GFVIFSVLGY MAHTLGVRIE DVA TEGPGL VFVVYPAAIA TMPASTFWAL IFFMMLATLG LDSSFGGSEA IITALSDEFP KIKRNRELFV AGLFSLYFVV GLAS CTQGG FYFFHLLDRY AAGYSILVAV FFEAIAVSWI YGTNRFSEDI RDMIGFPPGR YWQVCWRFVA PIFLLFITVY LLIGY EPLT YADYVYPSWA NALGWCIAGS SVVMIPAVAI FKLLSTPGSL RQRFTILTTP WRDQQLVPR

UniProtKB: Sodium-dependent dopamine transporter

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Macromolecule #2: 9D5 ANTIBODY, LIGHT CHAIN

MacromoleculeName: 9D5 ANTIBODY, LIGHT CHAIN / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.840607 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MDFQVQIFSF LLISASVAMS RGENVLTQSP AIMSTSPGEK VTMTCRASSS VGSSYLHWYQ QKSGASPKLW IYSTSNLASG VPARFSGSG SGTSYSLTIS SVEAEDAATY YCQQFSGYPL TFGSGTKLEM KRADAAPTVS IFPPSSEQLT SGGASVVCFL N NFYPKDIN ...String:
MDFQVQIFSF LLISASVAMS RGENVLTQSP AIMSTSPGEK VTMTCRASSS VGSSYLHWYQ QKSGASPKLW IYSTSNLASG VPARFSGSG SGTSYSLTIS SVEAEDAATY YCQQFSGYPL TFGSGTKLEM KRADAAPTVS IFPPSSEQLT SGGASVVCFL N NFYPKDIN VKWKIDGSER QNGVLNSWTD QDSKDSTYSM SSTLTLTKDE YERHNSYTCE ATHKTSTSPI VKSFNRNEC

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Macromolecule #3: 9D5 ANTIBODY, HEAVY CHAIN

MacromoleculeName: 9D5 ANTIBODY, HEAVY CHAIN / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.921338 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MNFGLRLVFL VLILKGVQCE VQLVESGGGL VKPGGSLKLS CAASGFTFSS YAMSWVRQSP EKRLEWVAEI SSGGRYIYYS DTVTGRFTI SRDNARNILH LEMSSLRSED TAMYYCARGE VRQRGFDYWG QGTTLTVSSA KTTAPSVYPL APVCGDTTGS S VTLGCLVK ...String:
MNFGLRLVFL VLILKGVQCE VQLVESGGGL VKPGGSLKLS CAASGFTFSS YAMSWVRQSP EKRLEWVAEI SSGGRYIYYS DTVTGRFTI SRDNARNILH LEMSSLRSED TAMYYCARGE VRQRGFDYWG QGTTLTVSSA KTTAPSVYPL APVCGDTTGS S VTLGCLVK GYFPEPVTLT WNSGSLSSGV HTFPAVLQSD LYTLSSSVTV TSSTWPSQSI TCNVAHPASS TKVDKKIEPR GP

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Macromolecule #4: N-[3-(6-chloranyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-2-yl)propy...

MacromoleculeName: N-[3-(6-chloranyl-1,3,4,9-tetrahydropyrido[3,4-b]indol-2-yl)propyl]butanamide
type: ligand / ID: 4 / Number of copies: 1 / Formula: A1H8F
Molecular weightTheoretical: 333.856 Da

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: TRIS-HYDROXYMETHYL-METHYL-AMMONIUM

MacromoleculeName: TRIS-HYDROXYMETHYL-METHYL-AMMONIUM / type: ligand / ID: 6 / Number of copies: 1 / Formula: 144
Molecular weightTheoretical: 122.143 Da
Chemical component information

ChemComp-144:
TRIS-HYDROXYMETHYL-METHYL-AMMONIUM

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Macromolecule #7: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 7 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #8: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 8 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #9: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2.0 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMNaClsodium chloride
20.0 mMTris-HCltris hydrochloride
0.01 (w/v) %LMNGLauryl Maltose Neopentyl Glycol
0.001 (w/v) %CHScholesterol-hemisuccinate
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 14624 / Average electron dose: 61.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4m48

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 147413
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

4m48


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-9euo:
Outward-open structure of Drosophila dopamine transporter bound to an atypical non-competitive inhibitor

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