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- EMDB-41081: Cryo-EM structure of human Anion Exchanger 1 bound to Dipyridamole -

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Basic information

Entry
Database: EMDB / ID: EMD-41081
TitleCryo-EM structure of human Anion Exchanger 1 bound to Dipyridamole
Map datastructure of human Anion Exchanger 1 bound to Dipyridamole
Sample
  • Complex: Dimeric anion exchanger 1 (SLC4A1)
    • Protein or peptide: Band 3 anion transport protein
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 2-[[2-[bis(2-hydroxyethyl)amino]-4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-6-yl]-(2-hydroxyethyl)amino]ethanol
  • Ligand: water
KeywordsTransmembrane / TRANSPORT PROTEIN
Function / homology
Function and homology information


pH elevation / Defective SLC4A1 causes hereditary spherocytosis type 4 (HSP4), distal renal tubular acidosis (dRTA) and dRTA with hemolytic anemia (dRTA-HA) / negative regulation of urine volume / Bicarbonate transporters / intracellular monoatomic ion homeostasis / ankyrin-1 complex / plasma membrane phospholipid scrambling / monoatomic anion transmembrane transporter activity / chloride:bicarbonate antiporter activity / solute:inorganic anion antiporter activity ...pH elevation / Defective SLC4A1 causes hereditary spherocytosis type 4 (HSP4), distal renal tubular acidosis (dRTA) and dRTA with hemolytic anemia (dRTA-HA) / negative regulation of urine volume / Bicarbonate transporters / intracellular monoatomic ion homeostasis / ankyrin-1 complex / plasma membrane phospholipid scrambling / monoatomic anion transmembrane transporter activity / chloride:bicarbonate antiporter activity / solute:inorganic anion antiporter activity / bicarbonate transmembrane transporter activity / bicarbonate transport / monoatomic anion transport / chloride transport / chloride transmembrane transporter activity / erythrocyte development / negative regulation of glycolytic process through fructose-6-phosphate / ankyrin binding / hemoglobin binding / cortical cytoskeleton / protein-membrane adaptor activity / chloride transmembrane transport / protein localization to plasma membrane / regulation of intracellular pH / transmembrane transport / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / cytoplasmic side of plasma membrane / Z disc / blood coagulation / basolateral plasma membrane / blood microparticle / protein homodimerization activity / extracellular exosome / membrane / plasma membrane
Similarity search - Function
Anion exchange protein 1 / Anion exchange protein / Anion exchange, conserved site / Anion exchangers family signature 1. / Anion exchangers family signature 2. / Band 3 cytoplasmic domain / Band 3 cytoplasmic domain / Bicarbonate transporter, eukaryotic / Bicarbonate transporter-like, transmembrane domain / HCO3- transporter family / Phosphotransferase/anion transporter
Similarity search - Domain/homology
Band 3 anion transport protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsCapper MJ / Zilberg G / Mathiharan YK / Yang S / Stone AC / Wacker D
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM133504 United States
Other privateEdward Mallinckrodt, Jr Foundation Grant
Other privateMcKnight Endowment Fund for Neuroscience Scholarship
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Substrate binding and inhibition of the anion exchanger 1 transporter.
Authors: Michael J Capper / Shifan Yang / Alexander C Stone / Sezen Vatansever / Gregory Zilberg / Yamuna Kalyani Mathiharan / Raul Habib / Keino Hutchinson / Yihan Zhao / Avner Schlessinger / Mihaly ...Authors: Michael J Capper / Shifan Yang / Alexander C Stone / Sezen Vatansever / Gregory Zilberg / Yamuna Kalyani Mathiharan / Raul Habib / Keino Hutchinson / Yihan Zhao / Avner Schlessinger / Mihaly Mezei / Roman Osman / Bin Zhang / Daniel Wacker /
Abstract: Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO transport between lungs and tissues. Previous ...Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO transport between lungs and tissues. Previous studies characterized its role in erythrocyte structure and provided insight into transport regulation. However, key questions remain regarding substrate binding and transport, mechanisms of drug inhibition and modulation by membrane components. Here we present seven cryo-EM structures in apo, bicarbonate-bound and inhibitor-bound states. These, combined with uptake and computational studies, reveal important molecular features of substrate recognition and transport, and illuminate sterol binding sites, to elucidate distinct inhibitory mechanisms of research chemicals and prescription drugs. We further probe the substrate binding site via structure-based ligand screening, identifying an AE1 inhibitor. Together, our findings provide insight into mechanisms of solute carrier transport and inhibition.
History
DepositionJun 18, 2023-
Header (metadata) releaseSep 13, 2023-
Map releaseSep 13, 2023-
UpdateNov 1, 2023-
Current statusNov 1, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_41081.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationstructure of human Anion Exchanger 1 bound to Dipyridamole
Voxel sizeX=Y=Z: 1.076 Å
Density
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-0.49783635 - 1.185456
Average (Standard dev.)-0.0026466555 (±0.03184066)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 361.53598 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Additional Map

Fileemd_41081_additional_1.map
AnnotationAdditional Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half Map 1

Fileemd_41081_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half Map 2

Fileemd_41081_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Dimeric anion exchanger 1 (SLC4A1)

EntireName: Dimeric anion exchanger 1 (SLC4A1)
Components
  • Complex: Dimeric anion exchanger 1 (SLC4A1)
    • Protein or peptide: Band 3 anion transport protein
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 2-[[2-[bis(2-hydroxyethyl)amino]-4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-6-yl]-(2-hydroxyethyl)amino]ethanol
  • Ligand: water

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Supramolecule #1: Dimeric anion exchanger 1 (SLC4A1)

SupramoleculeName: Dimeric anion exchanger 1 (SLC4A1) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 203 KDa

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Macromolecule #1: Band 3 anion transport protein

MacromoleculeName: Band 3 anion transport protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 58.254105 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DDPLQQTGQL FGGLVRDIRR RYPYYLSDIT DAFSPQVLAA VIFIYFAALS PAITFGGLLG EKTRNQMGVS ELLISTAVQG ILFALLGAQ PLLVVGFSGP LLVFEEAFFS FCETNGLEYI VGRVWIGFWL ILLVVLVVAF EGSFLVRFIS RYTQEIFSFL I SLIFIYET ...String:
DDPLQQTGQL FGGLVRDIRR RYPYYLSDIT DAFSPQVLAA VIFIYFAALS PAITFGGLLG EKTRNQMGVS ELLISTAVQG ILFALLGAQ PLLVVGFSGP LLVFEEAFFS FCETNGLEYI VGRVWIGFWL ILLVVLVVAF EGSFLVRFIS RYTQEIFSFL I SLIFIYET FSKLIKIFQD HPLQKTYNYN VLMVPKPQGP LPNTALLSLV LMAGTFFFAM MLRKFKNSSY FPGKLRRVIG DF GVPISIL IMVLVDFFIQ DTYTQKLSVP DGFKVSNSSA RGWVIHPLGL RSEFPIWMMF ASALPALLVF ILIFLESQIT TLI VSKPER KMVKGSGFHL DLLLVVGMGG VAALFGMPWL SATTVRSVTH ANALTVMGKA STPGAAAQIQ EVKEQRISGL LVAV LVGLS ILMEPILSRI PLAVLFGIFL YMGVTSLSGI QLFDRILLLF KPPKYHPDVP YVKRVKTWRM HLFTGIQIIC LAVLW VVKS TPASLALPFV LILTVPLRRV LLPLIFRNVE LQCLDADDA

UniProtKB: Band 3 anion transport protein

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Macromolecule #3: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 8 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #4: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 4 / Number of copies: 2 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM / Phosphatidylcholine

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Macromolecule #5: 2-[[2-[bis(2-hydroxyethyl)amino]-4,8-di(piperidin-1-yl)pyrimido[5...

MacromoleculeName: 2-[[2-[bis(2-hydroxyethyl)amino]-4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-6-yl]-(2-hydroxyethyl)amino]ethanol
type: ligand / ID: 5 / Number of copies: 2 / Formula: H9F
Molecular weightTheoretical: 504.626 Da
Chemical component information

ChemComp-H9F:
2-[[2-[bis(2-hydroxyethyl)amino]-4,8-di(piperidin-1-yl)pyrimido[5,4-d]pyrimidin-6-yl]-(2-hydroxyethyl)amino]ethanol / medication, inhibitor*YM / Dipyridamole

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 36 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration4.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHEPES
100.0 mMsodium chloride
0.001 % (w/v)LMNG
0.0001 % (w/v)CHS
0.0001 % (w/v)GDN
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 285 K / Instrument: LEICA EM GP
Details: Blot force 3 for 3-5 seconds was used and subsequent grids were screened for ice thickness prior to data collection.
DetailsThe sample was monodisperse following gel filtration and immediately concentrated for grid preparation

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 64000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4570 / Average electron dose: 51.69 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2499027
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
Details: Multiple rounds of 3D classification to remove particles
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 270791

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Atomic model buiding 1

Initial modelPDB ID:

Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL
Output model

PDB-8t6u:
Cryo-EM structure of human Anion Exchanger 1 bound to Dipyridamole

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