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- EMDB-23545: Cryo-EM structure of the wild-type human serotonin transporter co... -

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Basic information

Entry
Database: EMDB / ID: EMD-23545
TitleCryo-EM structure of the wild-type human serotonin transporter complexed with vilazodone, imipramine and 15B8 Fab
Map dataCryo-EM structure of the wild-type human serotonin transporter complexed with vilazodone, imipramine and 15B8 Fab
Sample
  • Complex: serotonin transporter 8B6 Fab complex
    • Complex: serotonin transporter
      • Protein or peptide: Sodium-dependent serotonin transporter
    • Complex: 8B6 antibody
      • Protein or peptide: heavy chain antibody fragment
      • Protein or peptide: light chain antibody fragment
  • Ligand: 3-(5H-DIBENZO[B,F]AZEPIN-5-YL)-N,N-DIMETHYLPROPAN-1-AMINE
  • Ligand: 5-{4-[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl}-1-benzofuran-2-carboxamide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


negative regulation of cerebellar granule cell precursor proliferation / regulation of thalamus size / Serotonin clearance from the synaptic cleft / serotonergic synapse / positive regulation of serotonin secretion / cocaine binding / serotonin:sodium:chloride symporter activity / sperm ejaculation / neurotransmitter transmembrane transporter activity / negative regulation of synaptic transmission, dopaminergic ...negative regulation of cerebellar granule cell precursor proliferation / regulation of thalamus size / Serotonin clearance from the synaptic cleft / serotonergic synapse / positive regulation of serotonin secretion / cocaine binding / serotonin:sodium:chloride symporter activity / sperm ejaculation / neurotransmitter transmembrane transporter activity / negative regulation of synaptic transmission, dopaminergic / monoamine transmembrane transporter activity / serotonin uptake / monoamine transport / enteric nervous system development / negative regulation of organ growth / cellular response to cGMP / sodium ion binding / serotonin binding / neurotransmitter transport / vasoconstriction / brain morphogenesis / antiporter activity / syntaxin-1 binding / nitric-oxide synthase binding / social behavior / membrane depolarization / negative regulation of neuron differentiation / sodium ion transmembrane transport / endomembrane system / positive regulation of cell cycle / monoatomic cation channel activity / cellular response to retinoic acid / response to nutrient / memory / response to toxic substance / platelet aggregation / circadian rhythm / actin filament binding / integrin binding / response to estradiol / presynaptic membrane / postsynaptic membrane / response to hypoxia / endosome membrane / neuron projection / response to xenobiotic stimulus / membrane raft / focal adhesion / synapse / positive regulation of gene expression / identical protein binding / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter, serotonin, N-terminal / Serotonin (5-HT) neurotransmitter transporter, N-terminus / 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 serotonin transporter
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsYang D / Kalenderoglou IE / Gouaux E / Coleman JA / Loland CJ
Funding support United States, Denmark, 7 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)5R37MH070039 United States
Howard Hughes Medical Institute (HHMI) United States
Danish Council for Independent Research7016-00272A Denmark
LundbeckfondenR344-2020-1020 Denmark
Novo Nordisk FoundationNNF19OC0058496 Denmark
The Carlsberg FoundationCF19-0527 Denmark
The Carlsberg FoundationCF20-0345 Denmark
CitationJournal: Nat Commun / Year: 2021
Title: The antidepressant drug vilazodone is an allosteric inhibitor of the serotonin transporter.
Authors: Per Plenge / Dongxue Yang / Kristine Salomon / Louise Laursen / Iris E Kalenderoglou / Amy H Newman / Eric Gouaux / Jonathan A Coleman / Claus J Loland /
Abstract: Depression is a common mental disorder. The standard medical treatment is the selective serotonin reuptake inhibitors (SSRIs). All characterized SSRIs are competitive inhibitors of the serotonin ...Depression is a common mental disorder. The standard medical treatment is the selective serotonin reuptake inhibitors (SSRIs). All characterized SSRIs are competitive inhibitors of the serotonin transporter (SERT). A non-competitive inhibitor may produce a more favorable therapeutic profile. Vilazodone is an antidepressant with limited information on its molecular interactions with SERT. Here we use molecular pharmacology and cryo-EM structural elucidation to characterize vilazodone binding to SERT. We find that it exhibits non-competitive inhibition of serotonin uptake and impedes dissociation of [H]imipramine at low nanomolar concentrations. Our SERT structure with bound imipramine and vilazodone reveals a unique binding pocket for vilazodone, expanding the boundaries of the extracellular vestibule. Characterization of the binding site is substantiated with molecular dynamics simulations and systematic mutagenesis of interacting residues resulting in decreased vilazodone binding to the allosteric site. Our findings underline the versatility of SERT allosteric ligands and describe the unique binding characteristics of vilazodone.
History
DepositionMar 1, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateSep 1, 2021-
Current statusSep 1, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.27
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7lwd
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23545.png

Downloads & links

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Map

FileDownload / File: emd_23545.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the wild-type human serotonin transporter complexed with vilazodone, imipramine and 15B8 Fab
Voxel sizeX=Y=Z: 0.648 Å
Density
Contour LevelBy AUTHOR: 0.27 / Movie #1: 0.27
Minimum - Maximum-1.620075 - 2.3069892
Average (Standard dev.)0.0018927015 (±0.040919334)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6480.6480.648
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z259.200259.200259.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-1.6202.3070.002

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Supplemental data

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Mask #1

Fileemd_23545_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_23545_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap1

Fileemd_23545_half_map_1.map
Annotationhalfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap2

Fileemd_23545_half_map_2.map
Annotationhalfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : serotonin transporter 8B6 Fab complex

EntireName: serotonin transporter 8B6 Fab complex
Components
  • Complex: serotonin transporter 8B6 Fab complex
    • Complex: serotonin transporter
      • Protein or peptide: Sodium-dependent serotonin transporter
    • Complex: 8B6 antibody
      • Protein or peptide: heavy chain antibody fragment
      • Protein or peptide: light chain antibody fragment
  • Ligand: 3-(5H-DIBENZO[B,F]AZEPIN-5-YL)-N,N-DIMETHYLPROPAN-1-AMINE
  • Ligand: 5-{4-[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl}-1-benzofuran-2-carboxamide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: serotonin transporter 8B6 Fab complex

SupramoleculeName: serotonin transporter 8B6 Fab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 105 KDa

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Supramolecule #2: serotonin transporter

SupramoleculeName: serotonin transporter / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: 8B6 antibody

SupramoleculeName: 8B6 antibody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Sodium-dependent serotonin transporter

MacromoleculeName: Sodium-dependent serotonin transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 61.062129 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GERETWGKKV DFLLSVIGYA VDLGNVWRFP YICYQNGGGA FLLPYTIMAI FGGIPLFYME LALGQYHRNG CISIWRKICP IFKGIGYAI CIIAFYIASY YNTIMAWALY YLISSFTDQL PWTSCKNSWN TGNCTNYFSE DNITWTLHST SPAEEFYTRH V LQIHRSKG ...String:
GERETWGKKV DFLLSVIGYA VDLGNVWRFP YICYQNGGGA FLLPYTIMAI FGGIPLFYME LALGQYHRNG CISIWRKICP IFKGIGYAI CIIAFYIASY YNTIMAWALY YLISSFTDQL PWTSCKNSWN TGNCTNYFSE DNITWTLHST SPAEEFYTRH V LQIHRSKG LQDLGGISWQ LALCIMLIFT VIYFSIWKGV KTSGKVVWVT ATFPYIILSV LLVRGATLPG AWRGVLFYLK PN WQKLLET GVWIDAAAQI FFSLGPGFGV LLAFASYNKF NNNCYQDALV TSVVNCMTSF VSGFVIFTVL GYMAEMRNED VSE VAKDAG PSLLFITYAE AIANMPASTF FAIIFFLMLI TLGLDSTFAG LEGVITAVLD EFPHVWAKRR ERFVLAVVIT CFFG SLVTL TFGGAYVVKL LEEYATGPAV LTVALIEAVA VSWFYGITQF CRDVKEMLGF SPGWFWRICW VAISPLFLLF IICSF LMSP PQLRLFQYNY PYWSIILGYC IGTSSFICIP TYIAYRLIIT PGTFKERIIK SITPETP

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Macromolecule #2: heavy chain antibody fragment

MacromoleculeName: heavy chain antibody fragment / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 12.980533 KDa
SequenceString:
QVQLQQSGPE LVKLGASVRI SCKASGYRFS YSWMNWVKQR PGKGLEWIGR IYPGDGDTKY SGKFKGKATL TADKSSSTVY MQLSSLTSE DSAVYFCARS AYGSEGFAMD YWGQGTSVT

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Macromolecule #3: light chain antibody fragment

MacromoleculeName: light chain antibody fragment / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.776065 KDa
SequenceString:
DIVLTQSPAS LAVSLGQRAT ISCRASESVD NYGISFLNWF QQKPGQPPKL LIYAASNQGS GVPARFSGSG SGTYFSLNIH PMEEDDTAV YFCQQTKGVS WTFGGGTKVE I

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Macromolecule #4: 3-(5H-DIBENZO[B,F]AZEPIN-5-YL)-N,N-DIMETHYLPROPAN-1-AMINE

MacromoleculeName: 3-(5H-DIBENZO[B,F]AZEPIN-5-YL)-N,N-DIMETHYLPROPAN-1-AMINE
type: ligand / ID: 4 / Number of copies: 1 / Formula: IXX
Molecular weightTheoretical: 280.407 Da
Chemical component information

ChemComp-IXX:
3-(5H-DIBENZO[B,F]AZEPIN-5-YL)-N,N-DIMETHYLPROPAN-1-AMINE / antidepressant*YM / Imipramine

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Macromolecule #5: 5-{4-[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl}-1-benzofuran...

MacromoleculeName: 5-{4-[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl}-1-benzofuran-2-carboxamide
type: ligand / ID: 5 / Number of copies: 1 / Formula: YG7
Molecular weightTheoretical: 441.525 Da
Chemical component information

ChemComp-YG7:
5-{4-[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl}-1-benzofuran-2-carboxamide / medication*YM / Vilazodone

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Macromolecule #6: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 6 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration4 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMTris
100.0 mMsodium chloride
1.0 mMDodecyl maltopyranoside
0.2 mMCholesteryl hemisuccinate
VitrificationCryogen name: ETHANE
Detailsmonodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 43.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: OTHER / Details: ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 415288
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6awn

RefinementProtocol: RIGID BODY FIT
Output model

PDB-7lwd:
Cryo-EM structure of the wild-type human serotonin transporter complexed with vilazodone, imipramine and 15B8 Fab

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