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- PDB-6w2b: Anomalous bromine signal reveals the position of Br-paroxetine co... -

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Basic information

Entry
Database: PDB / ID: 6w2b
TitleAnomalous bromine signal reveals the position of Br-paroxetine complexed with the serotonin transporter at the central site
Components
  • 8B6 heavy chain antibody fragment
  • 8B6 light chain antibody fragment
  • Sodium-dependent serotonin transporter
KeywordsTRANSPORT PROTEIN / antidepressant / complex / transporter / antibody
Function / homology
Function and homology information


negative regulation of cerebellar granule cell precursor proliferation / regulation of thalamus size / Serotonin clearance from the synaptic cleft / positive regulation of serotonin secretion / serotonergic synapse / negative regulation of synaptic transmission, dopaminergic / cocaine binding / serotonin:sodium:chloride symporter activity / cellular response to cGMP / enteric nervous system development ...negative regulation of cerebellar granule cell precursor proliferation / regulation of thalamus size / Serotonin clearance from the synaptic cleft / positive regulation of serotonin secretion / serotonergic synapse / negative regulation of synaptic transmission, dopaminergic / cocaine binding / serotonin:sodium:chloride symporter activity / cellular response to cGMP / enteric nervous system development / negative regulation of organ growth / sodium ion binding / neurotransmitter transmembrane transporter activity / serotonin uptake / vasoconstriction / monoamine transmembrane transporter activity / monoamine transport / serotonin binding / brain morphogenesis / syntaxin-1 binding / antiporter activity / negative regulation of neuron differentiation / male mating behavior / neurotransmitter transport / nitric-oxide synthase binding / amino acid transport / conditioned place preference / membrane depolarization / monoatomic cation channel activity / behavioral response to cocaine / positive regulation of cell cycle / cellular response to retinoic acid / response to nutrient / endomembrane system / sodium ion transmembrane transport / circadian rhythm / response to toxic substance / platelet aggregation / memory / integrin binding / actin filament binding / response to estradiol / presynaptic membrane / postsynaptic membrane / response to hypoxia / neuron projection / endosome membrane / membrane raft / response to xenobiotic stimulus / 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
Br-paroxetine / Sodium-dependent serotonin transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 4.7 Å
AuthorsColeman, J.A. / Navratna, V. / Yang, D.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)5R37MH070039 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2020
Title: Chemical and structural investigation of the paroxetine-human serotonin transporter complex.
Authors: Jonathan A Coleman / Vikas Navratna / Daniele Antermite / Dongxue Yang / James A Bull / Eric Gouaux /
Abstract: Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally ...Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.
History
DepositionMar 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 25, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 15, 2020Group: 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description ..._chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.3Sep 23, 2020Group: Database references / Structure summary / Category: chem_comp / citation_author
Item: _chem_comp.pdbx_synonyms / _citation_author.identifier_ORCID
Revision 1.4Oct 18, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.5Nov 6, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium-dependent serotonin transporter
B: 8B6 heavy chain antibody fragment
C: 8B6 light chain antibody fragment
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,8875
Polymers110,2763
Non-polymers6112
Water00
1
A: Sodium-dependent serotonin transporter
hetero molecules

B: 8B6 heavy chain antibody fragment
C: 8B6 light chain antibody fragment


Theoretical massNumber of molelcules
Total (without water)110,8875
Polymers110,2763
Non-polymers6112
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation3_657-x+1,y,-z+5/21
Buried area6190 Å2
ΔGint-50 kcal/mol
Surface area41230 Å2
MethodPISA
Unit cell
Length a, b, c (Å)128.020, 161.910, 139.660
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number20
Space group name H-MC2221
Space group name HallC2c2
Symmetry operation#1: x,y,z
#2: x,-y,-z
#3: -x,y,-z+1/2
#4: -x,-y,z+1/2
#5: x+1/2,y+1/2,z
#6: x+1/2,-y+1/2,-z
#7: -x+1/2,y+1/2,-z+1/2
#8: -x+1/2,-y+1/2,z+1/2

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Components

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


Mass: 61703.789 Da / Num. of mol.: 1 / Fragment: UNP residues 76-618
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
#2: Antibody 8B6 heavy chain antibody fragment


Mass: 24853.619 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 8B6 light chain antibody fragment


Mass: 23718.217 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)
#4: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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
#5: Chemical ChemComp-RFS / Br-paroxetine / (3S,4R)-3-{[(2H-1,3-benzodioxol-5-yl)oxy]methyl}-4-(4-bromophenyl)piperidine


Mass: 390.271 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H20BrNO3 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.28 Å3/Da / Density % sol: 62.52 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop
Details: 50 mM Tris, pH 8.5, 20 mM sodium sulfate, 20 mM lithium chloride, 36% PEG400, 0.5% 6-aminohexanoic acid

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-C / Wavelength: 0.9184 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jun 30, 2019
RadiationMonochromator: cryo-cooled double crystal Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9184 Å / Relative weight: 1
ReflectionResolution: 4.69→30 Å / Num. obs: 14667 / % possible obs: 99.2 % / Redundancy: 6.8 % / Biso Wilson estimate: 330.18 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.136 / Net I/σ(I): 5.51
Reflection shellResolution: 4.69→4.82 Å / Rmerge(I) obs: 0.3393 / Num. unique obs: 1130 / CC1/2: 0.165

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Processing

Software
NameVersionClassification
Cootmodel building
PHENIX1.15.2_3472refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 6AWN

6awn


Resolution: 4.7→29.76 Å / SU ML: 0.9316 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 43.3396
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2933 715 4.93 %
Rwork0.2594 13797 -
obs0.2611 14512 99.83 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 362.07 Å2
Refinement stepCycle: LAST / Resolution: 4.7→29.76 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7599 0 68 0 7667
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00777891
X-RAY DIFFRACTIONf_angle_d0.993610774
X-RAY DIFFRACTIONf_chiral_restr0.22691218
X-RAY DIFFRACTIONf_plane_restr0.00661325
X-RAY DIFFRACTIONf_dihedral_angle_d17.20052734
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
4.7-5.060.38861450.35522731X-RAY DIFFRACTION99.9
5.06-5.570.38371450.34312780X-RAY DIFFRACTION99.86
5.57-6.370.35281390.31232758X-RAY DIFFRACTION99.93
6.37-80.27571450.29372774X-RAY DIFFRACTION99.93
8-29.760.27371410.22732754X-RAY DIFFRACTION99.52

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