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- EMDB-43128: Structure of the human dopamine transporter in complex with beta-... -

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Basic information

Entry
Database: EMDB / ID: EMD-43128
TitleStructure of the human dopamine transporter in complex with beta-CFT, MRS7292 and divalent zinc
Map data
Sample
  • Complex: Sodium dependent dopamine transporter in complex with inhibitor
    • Protein or peptide: x 1 types
  • Ligand: x 13 types
KeywordsTransport / Membrane / Inhibitor / MEMBRANE PROTEIN
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
Sodium-dependent dopamine transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsSrivastava DK / Gouaux E
Funding support United States, 2 items
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, 2023-
Header (metadata) releaseAug 7, 2024-
Map releaseAug 7, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43128.png

Downloads & links

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Map

FileDownload / File: emd_43128.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 384 pix.
= 285.312 Å		0.74 Å/pix.
x 384 pix.
= 285.312 Å		0.74 Å/pix.
x 384 pix.
= 285.312 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.743 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.24
Minimum - Maximum-0.79284257 - 1.2521849
Average (Standard dev.)0.00047445484 (±0.02397155)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 285.31198 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43128_msk_1.map
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AxesZYX

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Half map: #2

Fileemd_43128_half_map_1.map
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Half map: #1

Fileemd_43128_half_map_2.map
Projections & Slices
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Sample components

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Entire : Sodium dependent dopamine transporter in complex with inhibitor

EntireName: Sodium dependent dopamine transporter in complex with inhibitor
Components
  • Complex: Sodium dependent dopamine transporter in complex with inhibitor
    • Protein or peptide: Sodium-dependent dopamine transporter
  • Ligand: methyl (1R,2S,3S,5S)-3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate
  • Ligand: 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
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: DECANE
  • Ligand: PENTANE
  • Ligand: HEPTANE
  • Ligand: ZINC ION
  • Ligand: SODIUM ION
  • Ligand: DODECANE
  • Ligand: PENTADECANE
  • Ligand: N-OCTANE
  • Ligand: water

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Supramolecule #1: Sodium dependent dopamine transporter in complex with inhibitor

SupramoleculeName: Sodium dependent dopamine transporter in complex with inhibitor
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 63 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: Homo sapiens (human)
Molecular weightTheoretical: 63.3845 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPGGRMAQDR ETWGKKIDFL LSVIGFAVDL ANVWRFPYLC YKNGGGAFLV PYLLFMVIAG MPLFYMELAL GQFNREGAAG VWKICPILK GVGFTVILIS LYVGFFYNVI IAWALHYLFS SFTTELPWIH CNNSWNSPNC SDAHPGDSSG DSSGLNDTFG T TPAAEYFE ...String:
GPGGRMAQDR ETWGKKIDFL LSVIGFAVDL ANVWRFPYLC YKNGGGAFLV PYLLFMVIAG MPLFYMELAL GQFNREGAAG VWKICPILK GVGFTVILIS LYVGFFYNVI IAWALHYLFS SFTTELPWIH CNNSWNSPNC SDAHPGDSSG DSSGLNDTFG T TPAAEYFE RGVLHLHQSH GIDDLGPPRW QLTACLVLVY VLLYFSLWKG VKTSGKVVWI TATMPYVVLT ALLLRGVTLP GA IDGIRAY LSVDFYRLCE ASVWIDAATQ VCFSLGVGFG VLIAFSSYNK FTNNCYRDAI VTTSINSLTS FSSGFVVFSF LGY MAQKHS VPIGDVAKDG PGLIFIIYPE AIATLPLSSA WAVVFFIMLL TLGIDSAMGG MESVITGLID EFQLLHRHRE LFTL FIVLA TFLLSLFCVT NGGIYVFTLL DHFAAGTSIL FGVLIEAIGV AWFYGVGQFS DDIQQMTGQR PSLYWRLCWK LVSPC FLLF VVVVSIVTFR PPHYGAYIFP DWANALGWVI ATSSMAMVPI YAAYKFCSLP GSFREKLAYA IAPEKDRELV DRGEVR QFT LRHWLKVI

UniProtKB: Sodium-dependent dopamine transporter

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Macromolecule #2: methyl (1R,2S,3S,5S)-3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2...

MacromoleculeName: methyl (1R,2S,3S,5S)-3-(4-fluorophenyl)-8-methyl-8-azabicyclo[3.2.1]octane-2-carboxylate
type: ligand / ID: 2 / Number of copies: 1 / Formula: 42L
Molecular weightTheoretical: 277.334 Da
Chemical component information

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

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Macromolecule #3: methyl (1S,2R,3S,4R,5R)-4-{2-[(5-chlorothiophen-2-yl)ethynyl]-6-(...

MacromoleculeName: 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
type: ligand / ID: 3 / Number of copies: 1 / Formula: A1AAF
Molecular weightTheoretical: 459.906 Da

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 5 / Number of copies: 4 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #6: DECANE

MacromoleculeName: DECANE / type: ligand / ID: 6 / Number of copies: 8 / Formula: D10
Molecular weightTheoretical: 142.282 Da
Chemical component information

ChemComp-D10:
DECANE

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Macromolecule #7: PENTANE

MacromoleculeName: PENTANE / type: ligand / ID: 7 / Number of copies: 7 / Formula: LNK
Molecular weightTheoretical: 72.149 Da
Chemical component information

ChemComp-LNK:
PENTANE

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Macromolecule #8: HEPTANE

MacromoleculeName: HEPTANE / type: ligand / ID: 8 / Number of copies: 8 / Formula: HP6
Molecular weightTheoretical: 100.202 Da
Chemical component information

ChemComp-HP6:
HEPTANE

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Macromolecule #9: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #10: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 10 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

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Macromolecule #11: DODECANE

MacromoleculeName: DODECANE / type: ligand / ID: 11 / Number of copies: 6 / Formula: D12
Molecular weightTheoretical: 170.335 Da
Chemical component information

ChemComp-D12:
DODECANE

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Macromolecule #12: PENTADECANE

MacromoleculeName: PENTADECANE / type: ligand / ID: 12 / Number of copies: 1 / Formula: MYS
Molecular weightTheoretical: 212.415 Da
Chemical component information

ChemComp-MYS:
PENTADECANE

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Macromolecule #13: N-OCTANE

MacromoleculeName: N-OCTANE / type: ligand / ID: 13 / Number of copies: 2 / Formula: OCT
Molecular weightTheoretical: 114.229 Da
Chemical component information

ChemComp-OCT:
N-OCTANE

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Macromolecule #14: water

MacromoleculeName: water / type: ligand / ID: 14 / Number of copies: 10 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration7.0 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
50.0 mMHEPESHEPES
200.0 mMNaClSodium Chloride
0.02 %DigitoninDetergent
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288.15 K / Instrument: FEI VITROBOT MARK IV
Detailsmonodisperse sample

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 6 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 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: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsFalcon 4i
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 177494
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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