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- PDB-8xb4: Structure of apo state of the human Norepinephrine Transporter -

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Basic information

Entry
Database: PDB / ID: 8xb4
TitleStructure of apo state of the human Norepinephrine Transporter
ComponentsGFP-MBP-solute carrier family 6 member 2
KeywordsTRANSPORT PROTEIN
Function / homology
Function and homology information


neurotransmitter:sodium symporter activity / Defective SLC6A2 causes orthostatic intolerance (OI) / norepinephrine uptake / norepinephrine:sodium symporter activity / dopamine:sodium symporter activity / norepinephrine transport / neurotransmitter transmembrane transporter activity / monoamine transmembrane transporter activity / monoamine transport / Na+/Cl- dependent neurotransmitter transporters ...neurotransmitter:sodium symporter activity / Defective SLC6A2 causes orthostatic intolerance (OI) / norepinephrine uptake / norepinephrine:sodium symporter activity / dopamine:sodium symporter activity / norepinephrine transport / neurotransmitter transmembrane transporter activity / monoamine transmembrane transporter activity / monoamine transport / Na+/Cl- dependent neurotransmitter transporters / neurotransmitter transport / amino acid transport / response to pain / dopamine uptake involved in synaptic transmission / neuronal cell body membrane / detection of maltose stimulus / maltose transport complex / beta-tubulin binding / carbohydrate transport / maltose binding / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / alpha-tubulin binding / sodium ion transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / neuron cellular homeostasis / presynaptic membrane / actin binding / outer membrane-bounded periplasmic space / chemical synaptic transmission / periplasmic space / response to xenobiotic stimulus / axon / DNA damage response / cell surface / membrane / metal ion binding / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter, noradrenaline / 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. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. ...Sodium:neurotransmitter symporter, noradrenaline / 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. / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Bacterial extracellular solute-binding protein
Similarity search - Domain/homology
Maltose/maltodextrin-binding periplasmic protein / Sodium-dependent noradrenaline transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsWu, J.X. / Ji, W.M.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32371266 China
CitationJournal: Nature / Year: 2024
Title: Substrate binding and inhibition mechanism of norepinephrine transporter.
Authors: Wenming Ji / Anran Miao / Kai Liang / Jiameng Liu / Yuhan Qi / Yue Zhou / Xinli Duan / Jixue Sun / Lipeng Lai / Jing-Xiang Wu /
Abstract: Norepinephrine transporter (NET; encoded by SLC6A2) reuptakes the majority of the released noradrenaline back to the presynaptic terminals, thereby affecting the synaptic noradrenaline level. Genetic ...Norepinephrine transporter (NET; encoded by SLC6A2) reuptakes the majority of the released noradrenaline back to the presynaptic terminals, thereby affecting the synaptic noradrenaline level. Genetic mutations and dysregulation of NET are associated with a spectrum of neurological conditions in humans, making NET an important therapeutic target. However, the structure and mechanism of NET remain unclear. Here we provide cryogenic electron microscopy structures of the human NET (hNET) in three functional states-the apo state, and in states bound to the substrate meta-iodobenzylguanidine (MIBG) or the orthosteric inhibitor radafaxine. These structures were captured in an inward-facing conformation, with a tightly sealed extracellular gate and an open intracellular gate. The substrate MIBG binds at the centre of hNET. Radafaxine also occupies the substrate-binding site and might block the structural transition of hNET for inhibition. These structures provide insights into the mechanism of substrate recognition and orthosteric inhibition of hNET.
History
DepositionDec 5, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 28, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.2Sep 18, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.3Sep 25, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GFP-MBP-solute carrier family 6 member 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)138,9182
Polymers138,6971
Non-polymers2211
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area170 Å2
ΔGint2 kcal/mol
Surface area22180 Å2

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Components

#1: Protein GFP-MBP-solute carrier family 6 member 2 / MMBP / Maltodextrin-binding protein / Maltose-binding protein / MBP / Solute carrier family 6 member 2


Mass: 138697.016 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human), (gene. exp.) Escherichia coli K-12 (bacteria)
Gene: SLC6A2, malE / Strain: K-12 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P0AEX9, UniProt: P23975
#2: 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: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
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
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

ComponentName: NET / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 52 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.16_3549: / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 194295 / Symmetry type: POINT
Atomic model buildingDetails: P23975 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044484
ELECTRON MICROSCOPYf_angle_d0.5646118
ELECTRON MICROSCOPYf_dihedral_angle_d4.0432499
ELECTRON MICROSCOPYf_chiral_restr0.043686
ELECTRON MICROSCOPYf_plane_restr0.005743

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