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- PDB-7lwd: Cryo-EM structure of the wild-type human serotonin transporter co... -

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Basic information

Entry
Database: PDB / ID: 7lwd
TitleCryo-EM structure of the wild-type human serotonin transporter complexed with vilazodone, imipramine and 15B8 Fab
Components
  • Sodium-dependent serotonin transporter
  • heavy chain antibody fragment
  • light chain antibody fragment
KeywordsTRANSPORT PROTEIN/Immune System / antidepressant / complex / transporter / antibody / TRANSPORT PROTEIN / TRANSPORT PROTEIN-Immune System complex
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 / sperm ejaculation / serotonin:sodium:chloride symporter activity / 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 / sperm ejaculation / serotonin:sodium:chloride symporter activity / neurotransmitter transmembrane transporter activity / negative regulation of synaptic transmission, dopaminergic / serotonin uptake / enteric nervous system development / monoamine transmembrane transporter activity / cellular response to cGMP / monoamine transport / negative regulation of organ growth / sodium ion binding / serotonin binding / vasoconstriction / neurotransmitter transport / brain morphogenesis / antiporter activity / syntaxin-1 binding / nitric-oxide synthase binding / membrane depolarization / social behavior / sodium ion transmembrane transport / negative regulation of neuron differentiation / endomembrane system / positive regulation of cell cycle / cellular response to retinoic acid / monoatomic cation channel activity / 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
Chem-IXX / Chem-YG7 / Sodium-dependent serotonin transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsYang, D. / Kalenderoglou, I.E. / Gouaux, E. / Coleman, J.A. / Loland, C.J.
Funding support United States, Denmark, 7items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 1, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Sodium-dependent serotonin transporter
H: heavy chain antibody fragment
L: light chain antibody fragment
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,7626
Polymers85,8193
Non-polymers9433
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Antibody , 2 types, 2 molecules HL

#2: Antibody heavy chain antibody fragment


Mass: 12980.533 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Cell line: Hybridoma
#3: Antibody light chain antibody fragment


Mass: 11776.065 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Mus musculus (house mouse) / Cell line: Hybridoma

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Protein / Sugars , 2 types, 2 molecules A

#1: Protein Sodium-dependent serotonin transporter / SERT / 5HT transporter / 5HTT / Solute carrier family 6 member 4


Mass: 61062.129 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A4, HTT, SERT / Cell line (production host): Hek293 GnTi- / Production host: Homo sapiens (human) / References: UniProt: P31645
#6: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 2 types, 2 molecules

#4: Chemical ChemComp-IXX / 3-(5H-DIBENZO[B,F]AZEPIN-5-YL)-N,N-DIMETHYLPROPAN-1-AMINE / 5-(3-(dimethylamino)propyl)-10,11-dihydro-5H-dibenz[b,f]azepine;10,11-Dehydroimipramine;Depramine / Imipramine


Mass: 280.407 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C19H24N2 / Comment: antidepressant*YM
#5: Chemical ChemComp-YG7 / 5-{4-[4-(5-cyano-1H-indol-3-yl)butyl]piperazin-1-yl}-1-benzofuran-2-carboxamide / Vilazodone


Mass: 441.525 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H27N5O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM

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Details

Has ligand of interestY

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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

Component
IDNameTypeEntity IDParent-IDSource
1serotonin transporter 8B6 Fab complexCOMPLEX#1-#30MULTIPLE SOURCES
2serotonin transporterCOMPLEX#11RECOMBINANT
38B6 antibodyCOMPLEX#2-#31NATURAL
Molecular weightValue: 0.105 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMTris1
2100 mMsodium chloride1
31 mMDodecyl maltopyranoside1
40.2 mMCholesteryl hemisuccinate1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 43 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameCategory
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cisTEMfinal Euler assignment
12RELIONclassification
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 415288 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6AWN

6awn

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036353
ELECTRON MICROSCOPYf_angle_d0.5558653
ELECTRON MICROSCOPYf_dihedral_angle_d10.1083584
ELECTRON MICROSCOPYf_chiral_restr0.039938
ELECTRON MICROSCOPYf_plane_restr0.0041062

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