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- EMDB-26168: Cryo-EM structure of human Anion Exchanger 1 bound to Bicarbonate -

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Basic information

Entry
Database: EMDB / ID: EMD-26168
TitleCryo-EM structure of human Anion Exchanger 1 bound to Bicarbonate
Map dataCryo-EM structure of human Anion Exchanger 1 bound to Bicarbonate
Sample
  • Complex: Dimeric anion exchanger 1 (SLC4A1)
    • Protein or peptide: Band 3 anion transport protein
  • Ligand: BICARBONATE IONBicarbonate
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • 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: 3.37 Å
AuthorsCapper MJ / Mathiharan YK
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
DepositionFeb 11, 2022-
Header (metadata) releaseAug 16, 2023-
Map releaseAug 16, 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_26168.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of human Anion Exchanger 1 bound to Bicarbonate
Voxel sizeX=Y=Z: 1.076 Å
Density
Contour LevelBy AUTHOR: 0.28
Minimum - Maximum-0.3082428 - 1.0935637
Average (Standard dev.)-0.0014764618 (±0.027873868)
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: Sharpened map

Fileemd_26168_additional_1.map
AnnotationSharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Half Map 1

Fileemd_26168_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_26168_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: BICARBONATE IONBicarbonate
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • 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: 101.883859 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEELQDDYED MMEENLEQEE YEDPDIPESQ MEEPAAHDTE ATATDYHTTS HPGTHKVYVE LQELVMDEKN QELRWMEAAR WVQLEENLG ENGAWGRPHL SHLTFWSLLE LRRVFTKGTV LLDLQETSLA GVANQLLDRF IFEDQIRPQD REELLRALLL K HSHAGELE ...String:
MEELQDDYED MMEENLEQEE YEDPDIPESQ MEEPAAHDTE ATATDYHTTS HPGTHKVYVE LQELVMDEKN QELRWMEAAR WVQLEENLG ENGAWGRPHL SHLTFWSLLE LRRVFTKGTV LLDLQETSLA GVANQLLDRF IFEDQIRPQD REELLRALLL K HSHAGELE ALGGVKPAVL TRSGDPSQPL LPQHSSLETQ LFCEQGDGGT EGHSPSGILE KIPPDSEATL VLVGRADFLE QP VLGFVRL QEAAELEAVE LPVPIRFLFV LLGPEAPHID YTQLGRAAAT LMSERVFRID AYMAQSRGEL LHSLEGFLDC SLV LPPTDA PSEQALLSLV PVQRELLRRR YQSSPAKPDS SFYKGLDLNG GPDDPLQQTG QLFGGLVRDI RRRYPYYLSD ITDA FSPQV LAAVIFIYFA ALSPAITFGG LLGEKTRNQM GVSELLISTA VQGILFALLG AQPLLVVGFS GPLLVFEEAF FSFCE TNGL EYIVGRVWIG FWLILLVVLV VAFEGSFLVR FISRYTQEIF SFLISLIFIY ETFSKLIKIF QDHPLQKTYN YNVLMV PKP QGPLPNTALL SLVLMAGTFF FAMMLRKFKN SSYFPGKLRR VIGDFGVPIS ILIMVLVDFF IQDTYTQKLS VPDGFKV SN SSARGWVIHP LGLRSEFPIW MMFASALPAL LVFILIFLES QITTLIVSKP ERKMVKGSGF HLDLLLVVGM GGVAALFG M PWLSATTVRS VTHANALTVM GKASTPGAAA QIQEVKEQRI SGLLVAVLVG LSILMEPILS RIPLAVLFGI FLYMGVTSL SGIQLFDRIL LLFKPPKYHP DVPYVKRVKT WRMHLFTGIQ IICLAVLWVV KSTPASLALP FVLILTVPLR RVLLPLIFRN VELQCLDAD DAKATFDEEE GRDEYDEVAM PV

UniProtKB: Band 3 anion transport protein

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Macromolecule #3: BICARBONATE ION

MacromoleculeName: BICARBONATE ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: BCT
Molecular weightTheoretical: 61.017 Da
Chemical component information

ChemComp-BCT:
BICARBONATE ION / pH buffer*YM / Bicarbonate

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Macromolecule #4: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 4 / Number of copies: 7 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #5: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 5 / 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 #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 20 / 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

Concentration5 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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
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: 5256 / Average electron dose: 59.99 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2660401
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: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 173471
FSC plot (resolution estimation)

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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-7ty7:
Cryo-EM structure of human Anion Exchanger 1 bound to Bicarbonate

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