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

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Basic information

Entry
Database: PDB / ID: 7ty7
TitleCryo-EM structure of human Anion Exchanger 1 bound to Bicarbonate
ComponentsBand 3 anion transport protein
KeywordsTRANSPORT PROTEIN / Transmembrane
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 transport / bicarbonate transmembrane transporter activity / monoatomic anion transport / chloride transport / chloride transmembrane transporter activity / ankyrin binding / negative regulation of glycolytic process through fructose-6-phosphate / hemoglobin binding / cortical cytoskeleton / erythrocyte development / protein-membrane adaptor activity / chloride transmembrane transport / protein localization to plasma membrane / regulation of intracellular pH / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / transmembrane transport / Z disc / cytoplasmic side of plasma membrane / 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 / Phosphotransferase/anion transporter / Bicarbonate transporter, eukaryotic / Bicarbonate transporter-like, transmembrane domain / HCO3- transporter integral membrane domain
Similarity search - Domain/homology
BICARBONATE ION / CHOLESTEROL / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Band 3 anion transport protein
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsCapper, M.J. / Mathiharan, Y.K. / Yang, S. / Stone, A.C. / Wacker, D.
Funding support United States, 3items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Sep 20, 2023Group: Database references / Refinement description / Category: citation / citation_author / em_3d_fitting_list
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type
Revision 1.3Nov 1, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Band 3 anion transport protein
B: Band 3 anion transport protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)209,02515
Polymers203,7682
Non-polymers5,25813
Water36020
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: ASP / Beg label comp-ID: ASP / End auth comp-ID: ALA / End label comp-ID: ALA / Auth seq-ID: 369 - 891 / Label seq-ID: 369 - 891

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

NCS ensembles : (Details: Global NCS restraints between domains: 1 2)
NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-1, 0.000223, 0.000752), (-0.000223, -1, 0.000149), (0.000752, 0.000149, 1)361.365655, 361.545651, -0.177318262

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Components

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein Band 3 anion transport protein / Anion exchange protein 1 / AE 1 / Anion exchanger 1 / Solute carrier family 4 member 1


Mass: 101883.859 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC4A1, AE1, DI, EPB3 / Plasmid: pFB / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P02730
#2: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE

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Non-polymers , 4 types, 31 molecules

#3: Chemical ChemComp-BCT / BICARBONATE ION


Mass: 61.017 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: CHO3 / Feature type: SUBJECT OF INVESTIGATION / Comment: pH buffer*YM
#4: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C27H46O
#5: Chemical ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE


Mass: 790.145 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 20 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: Dimeric anion exchanger 1 (SLC4A1) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.203 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: Sf9 / Plasmid: pFB
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2100 mMsodium chloride1
30.001 % (w/v)LMNG1
40.0001 % (w/v)CHS1
50.0001 % (w/v)GDN1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The sample was monodisperse following gel filtration and immediately concentrated for grid preparation
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 285 K
Details: Blot force 3 for 3-5 seconds was used and subsequent grids were screened for ice thickness prior to data collection

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 64000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 59.99 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5256

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13REFMACservalcatmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2660401
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173471 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
Atomic model buildingPDB-ID: 4YZF

4yzf


Accession code: 4YZF / Source name: PDB / Type: experimental model
RefinementResolution: 3.37→129.12 Å / Cor.coef. Fo:Fc: 0.904 / WRfactor Rwork: 0.358 / Average fsc overall: 0.8257 / Average fsc work: 0.8257 / ESU R: 0.652
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3577 63361 -
all0.358 --
Rfree--0 %
obs--100 %
Solvent computationSolvent model: BABINET MODEL
Displacement parametersBiso mean: 120.692 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0020.0138745
ELECTRON MICROSCOPYr_bond_other_d0.0330.0178954
ELECTRON MICROSCOPYr_angle_refined_deg1.2641.63511906
ELECTRON MICROSCOPYr_angle_other_deg2.2121.56620573
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.95851028
ELECTRON MICROSCOPYr_dihedral_angle_2_deg28.61220.641359
ELECTRON MICROSCOPYr_dihedral_angle_3_deg13.971151426
ELECTRON MICROSCOPYr_dihedral_angle_4_deg13.9641546
ELECTRON MICROSCOPYr_chiral_restr0.0450.21166
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.029219
ELECTRON MICROSCOPYr_gen_planes_other0.0130.021967
ELECTRON MICROSCOPYr_nbd_refined0.1440.23962
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1830.216554
ELECTRON MICROSCOPYr_nbtor_refined0.150.28602
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0680.27584
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.10.2414
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_other0.040.24
ELECTRON MICROSCOPYr_mcbond_it3.75312.0924124
ELECTRON MICROSCOPYr_mcbond_other3.75212.094123
ELECTRON MICROSCOPYr_mcangle_it6.45418.1425148
ELECTRON MICROSCOPYr_mcangle_other6.45318.1445149
ELECTRON MICROSCOPYr_scbond_it3.6413.4264621
ELECTRON MICROSCOPYr_scbond_other3.6413.4294622
ELECTRON MICROSCOPYr_scangle_it6.44219.9436758
ELECTRON MICROSCOPYr_scangle_other6.44219.9466759
ELECTRON MICROSCOPYr_lrange_it11.28149.939740
ELECTRON MICROSCOPYr_lrange_other11.28149.959741
Refine LS restraints NCS

Ens-ID: 1 / Refine-ID: ELECTRON MICROSCOPY / Type: tight positional; tight thermal / Rms dev Biso : 0.36216 Å2 / Rms dev position: 0.02972 Å / Weight Biso : 0.5 / Weight position: 0.05

Dom-IDAuth asym-ID
1A
2B
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
3.37-3.4581.70247601.70247600.5881.702
3.458-3.5520.93644940.93644940.6790.936
3.552-3.6550.70544410.70544410.7360.705
3.655-3.7670.55243710.55243710.7880.552
3.767-3.8910.40841370.40841370.8230.408
3.891-4.0270.34540840.34540840.8530.345
4.027-4.1790.30439330.30439330.8770.304
4.179-4.350.29636910.29636910.8910.296
4.35-4.5430.30436150.30436150.9120.304
4.543-4.7640.32734310.32734310.9150.327
4.764-5.0220.33232820.33232820.9190.332
5.022-5.3260.32830730.32830730.9020.328
5.326-5.6930.3129180.3129180.8820.31
5.693-6.1480.30927240.30927240.8590.309
6.148-6.7330.34524590.34524590.8350.345
6.733-7.5250.36222670.36222670.8180.362
7.525-8.6850.3619840.3619840.8440.36
8.685-10.6240.30816720.30816720.9020.308
10.624-14.9750.26812930.26812930.9260.268
14.975-129.120.4487320.4487320.9590.448

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