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- EMDB-38209: Structural mechanism of substrate binding and inhibition of the h... -

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Basic information

Entry
Database: EMDB / ID: EMD-38209
TitleStructural mechanism of substrate binding and inhibition of the human Norepinephrine Transporter
Map data
Sample
  • Complex: NET
    • Protein or peptide: GFP-MBP-solute carrier family 6 member 2
  • Ligand: Iobenguane
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsmeta-iodobenzylguanidine / Radafaxin / neuroendocrine tumors / antidepression / TRANSPORT PROTEIN
Function / homology
Function and homology information


neurotransmitter:sodium symporter activity / Defective SLC6A2 causes orthostatic intolerance (OI) / norepinephrine uptake / norepinephrine:sodium symporter activity / norepinephrine transport / dopamine:sodium symporter activity / 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 / norepinephrine transport / dopamine:sodium symporter activity / neurotransmitter transmembrane transporter activity / monoamine transmembrane transporter activity / monoamine transport / Na+/Cl- dependent neurotransmitter transporters / neurotransmitter transport / neuronal cell body membrane / response to pain / dopamine uptake involved in synaptic transmission / detection of maltose stimulus / amino acid transport / beta-tubulin binding / maltose transport complex / carbohydrate transport / carbohydrate transmembrane transporter activity / maltose binding / alpha-tubulin binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / sodium ion transmembrane transport / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / neuron cellular homeostasis / synaptic vesicle membrane / presynaptic membrane / actin binding / outer membrane-bounded periplasmic space / chemical synaptic transmission / periplasmic space / response to xenobiotic stimulus / axon / DNA damage response / cell surface / metal ion binding / membrane / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsJi WM / Wu JX
Funding support China, 1 items
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, 2023-
Header (metadata) releaseJun 26, 2024-
Map releaseJun 26, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38209.png

Downloads & links

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Map

FileDownload / File: emd_38209.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-4.2468452 - 6.403534
Average (Standard dev.)-0.0020270608 (±0.11952751)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_38209_msk_1.map
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Half map: #1

Fileemd_38209_half_map_1.map
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Half map: #2

Fileemd_38209_half_map_2.map
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Sample components

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Entire : NET

EntireName: NET
Components
  • Complex: NET
    • Protein or peptide: GFP-MBP-solute carrier family 6 member 2
  • Ligand: Iobenguane
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: NET

SupramoleculeName: NET / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: GFP-MBP-solute carrier family 6 member 2

MacromoleculeName: GFP-MBP-solute carrier family 6 member 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 138.697016 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATIG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMP EGYVQERTIS FKDDGKYKTR AVVKFEGDTL VNRIELKGTD FKEDGNILGH KLEYNFNSHN VYITADKQKN G IKANFTVR ...String:
MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDATIG KLTLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKR HDFFKSAMP EGYVQERTIS FKDDGKYKTR AVVKFEGDTL VNRIELKGTD FKEDGNILGH KLEYNFNSHN VYITADKQKN G IKANFTVR HNVEGSGHHH HHHHHADRKA AVSHWQQSSG LVPRGSGWSH PQFEKGSGDY KDDDDKGSGW SHPQFEKGSG DG SVQLADH YQQNTPIGDG PVLLPDNHYL STQTKLSKDP NEKRDHMVLH EYVNAAGITG SMKIEEGKLV IWINGDKGYN GLA EVGKKF EKDTGIKVTV EHPDKLEEKF PQVAATGDGP DIIFWAHDRF GGYAQSGLLA EITPDKAFQD KLYPFTWDAV RYNG KLIAY PIAVEALSLI YNKDLLPNPP KTWEEIPALD KELKAKGKSA LMFNLQEPYF TWPLIAADGG YAFKYENGKY DIKDV GVDN AGAKAGLTFL VDLIKNKHMN ADTDYSIAEA AFNKGETAMT INGPWAWSNI DTSKVNYGVT VLPTFKGQPS KPFVGV LSA GINAASPNKE LAKEFLENYL LTDEGLEAVN KDKPLGAVAL KSYEEELVKD PRIAATMENA QKGEIMPNIP QMSAFWY AV RTAVINAASG RQTVDEALKD AQTGGLEVLF QGPEFQPRET WGKKIDFLLS VVGFAVDLAN VWRFPYLCYK NGGGAFLI P YTLFLIIAGM PLFYMELALG QYNREGAATV WKICPFFKGV GYAVILIALY VGFYYNVIIA WSLYYLFSSF TLNLPWTDC GHTWNSPNCT DPKLGGVAMP GAEDDVVGYS KYKFTPAAEF YERGVLHLHE SSGIHDIGLP QWQLLLCLMV VVIVLYFSLW KGVKTSGKV VWITATLPYF VLFVLLVHGV TLPGASNGIN AYLHIDFYRL KEATVWIDAA TQIFFSLGAG FGVLIAFASY N KFDNNCYR DALLTSSINC ITSFVSGFAI FSILGYMAHE HKVNIEDVAT EGAGLVFILY PEAISTLSGS TFWAVVFFVM LL ALGLDSS MGGMEAVITG LADDFQVLKR HRKLFTFGVT FSTFLLALFC ITKGGIYVLT LLDTFAAGTS ILFAVLMEAI GVS WFYGVD RFSNDIQQMM GFRPGLYWRL CWKFVSPAFL LFVVVVSIIN FKPLTYDDYI FPPWANWVGW GIALSSMVLV PIYV IYKFL STQGSLWERL AYGITPENEH HLVAQRDIRQ FQLQHWLAI

UniProtKB: Maltose/maltodextrin-binding periplasmic protein, Sodium-dependent noradrenaline transporter

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Macromolecule #2: Iobenguane

MacromoleculeName: Iobenguane / type: ligand / ID: 2 / Number of copies: 1 / Formula: YMN
Molecular weightTheoretical: 275.09 Da

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / 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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 207930
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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