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Substrate binding and inhibition of the anion exchanger 1 transporter

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Abstract

Anion exchanger 1 (AE1), a member of the solute carrier (SLC) family, is the primary bicarbonate transporter in erythrocytes, regulating pH levels and CO2 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.

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Fig. 1: Cryo-EM structure of full-length human AE1/SLC4A1.
Fig. 2: Structural insight into bicarbonate binding at AE1.
Fig. 3: Structure of AE1 bound to chemically and pharmacologically diverse inhibitors.
Fig. 4: Structure-based discovery of a chemical AE1 inhibitors.
Fig. 5: Model of AE1-mediated bicarbonate transport and diverse mechanisms of AE1 transport inhibition by pharmacologically different drugs.

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Data availability

Density maps and structure coordinates have been deposited in the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB): AE1-apo (EMD-26165 and PDB 7TY4), AE1–bicarbonate (EMD-26168 and PDB 7TY7), AE1–DIDS (EMD-41082 and PDB 8T6V), AE1–H2DIDS (EMD-26167 and PDB 7TY6), AE1–DEPC (EMD-26171 and PDB 7TYA), AE1–dipyridamole (EMD-41081 and PDB 8T6U), AE1–NIF (EMD-26169 and PDB 7TY8). Source data are provided with this paper.

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Acknowledgements

This work was supported by NIH grant GM133504, a Sloan Research Fellowship in Neuroscience, an Edward Mallinckrodt, Jr. Foundation Grant, a McKnight Foundation Scholars Award (all to D.W.), NIH T32 Training Grant GM062754 and DA053558 (G.Z.), R01 CA277794 (A.S. and K.H.), NIH grant U01AG046170 (B.Z.), NIH grant RF1AG057440 (B.Z.), NIH grant R01AG068030 (B.Z.) and NIH grants R01DK073681, R01DK067555 and R01DK061659 (R.O.). Some of this work was performed at the National Center for cryo-EM Access and Training (NCCAT) and the Simons Electron Microscopy Center located at the New York Structural Biology Center, supported by the NIH Common Fund Transformative High Resolution Cryo-Electron Microscopy program (U24 GM129539,) and by grants from the Simons Foundation (SF349247) and NY State Assembly. We further acknowledge cryo-EM resources at the National Resource for Automated Molecular Microscopy located at the New York Structural Biology Center, supported by grants from the Simons Foundation (SF349247), NYSTAR and the NIH National Institute of General Medical Sciences (GM103310) with additional support from Agouron Institute (F00316) and NIH (OD019994). For additional data collection we are also grateful to staff at the Laboratory for BioMolecular Structure (LBMS), which is supported by the DOE Office of Biological and Environmental Research (KP160711). This work was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai. We also thank J. F. Fay for help with initial data processing.

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Author notes

  1. These authors contributed equally: Michael J. Capper, Shifan Yang, Alexander C. Stone.

Authors and Affiliations

  1. Department of Pharmacological Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Michael J. Capper, Yamuna Kalyani Mathiharan, Raul Habib, Keino Hutchinson, Yihan Zhao, Avner Schlessinger, Mihaly Mezei, Roman Osman, Bin Zhang & Daniel Wacker

  2. Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Shifan Yang, Alexander C. Stone, Sezen Vatansever, Yihan Zhao & Bin Zhang

  3. Mount Sinai Center for Transformative Disease Modeling, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Shifan Yang, Alexander C. Stone, Sezen Vatansever & Bin Zhang

  4. Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Gregory Zilberg, Yamuna Kalyani Mathiharan, Bin Zhang & Daniel Wacker

  5. Department of Artificial Intelligence and Human Health, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Avner Schlessinger & Bin Zhang

  6. Icahn Genomics Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA

    Bin Zhang

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  1. Michael J. Capper
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Contributions

M.J.C. designed experiments, expressed and purified protein for grid freezing, collected data, refined structures and edited the manuscript. S.Y. and A.C.S. purified protein, prepared samples for grid freezing, established and performed functional assays and edited the manuscript. S.V. performed molecular docking calculations with help from Y.Z., and helped analyze the structures. G.Z. prepared grids for structure determination and assisted with data collection, processing and refinement. Y.K.M. helped with data processing and structure refinement. R.H. helped establish protein expression and purification. K.H. performed volume calculations. A.S. supervised docking and volume calculation experiments and helped write the paper. R.O. performed molecular simulations and SACP analysis of substrate binding with help from M.M. B.Z. contributed to the study design and supervised computational studies. D.W. designed experiments, analyzed the data, supervised the overall project and management and wrote the manuscript.

Corresponding author

Correspondence to Daniel Wacker.

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Nature Structural & Molecular Biology thanks Reinhart A. F. Reithmeier and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Extended data

Extended Data Fig. 1 Cryo-EM structure determination of AE1 complexes as exemplified by AE1-DIDS complex.

a, Analytical size exclusion chromatography and SDS-PAGE show monodisperse and pure protein of AE1-DIDS. Data were collected on 300 keV Krios, a representative micrograph is shown, and processed in cryoSPARC: particles were picked from motion corrected micrographs, subjected to 2D classification (representative classes are shown), followed by ab initio model building and 3D classification. After multiple rounds of 3D classification, the final particle stack was subjected to local CTF refinement followed by local refinement of the masked AE1 membrane domain with imposed C2 symmetry. Final map was obtained with GS-FSC indicating a resolution of 2.95 Å (AE1-DIDS) applying the 0.143 cutoff. Viewing direction distribution analysis (cryoSPARC) indicates sufficient coverage. An initial model was built in PHENIX, and then further refined in ServalCat for the generation of final maps and coordinates of mdAE1. Calculations in cryoSPARC indicate local resolutions of up to 2.5 Å around substrate and inhibitor binding sites. Viewing direction analysis indicates isotropic distribution of views in final particle stack. b, Superposition with other AE1 structures (AE1, PDB ID: 7TW2, dark red; AE1-Glycophorin complex, PDB ID: 7UZ3, lime green)7,8. Root mean square deviations of 0.997 Å (7TW2) and 0.372 Å (7UZ3) highlight similarity of protein conformation. Glycosylation modifications, cholesterols, lipids, waters, PIP2, and Glycophorin are shown in green, yellow, purple, red, grey, and teal, respectively. c, Cryo-EM structures allowed us to assign the complete extracellular surface including all Diego antigens (see Extended Data Table 2) with sidechains shown as spheres with yellow carbon atoms.

Source data

Extended Data Fig. 2 Cryo-EM Structure Determination of Apo AE1 and AE1-Bicarbonate complexes.

a, b, workflow of Apo AE1 (a) and AE1-Bicarbonate (b) data processing, showing representative 2D classes, followed by 2D classification, ab initio modeling and several rounds of 3D classification, with representative 3D classes shown. After multiple rounds of 3D classification, final particle stacks were subjected to CTF refinement followed by local refinement of the masked AE1 membrane domain with imposed C2 symmetry. GS-FSC calculations indicate global resolutions of 2.99 Å (Apo AE1) and 3.37 (AE1-Bicarbonate) applying the 0.143 cutoff. Viewing direction distribution analysis (cryoSPARC) indicates sufficient coverage. Initial models were built in PHENIX, and then further refined in ServalCat for the generation of final maps and coordinates. Calculations in cryoSPARC indicate local resolutions of lower than 3 Å around substrate and inhibitor binding sites.

Source data

Extended Data Fig. 3 Cryo-EM Structure Determination of AE1-H2DIDS and AE1-DEPC complexes.

a, b, workflow of AE1-H2DIDS (a) and AE1-DEPC (b) data processing, showing representative 2D classes, followed by 2D classification, ab initio modeling and several rounds of 3D classification, with representative 3D classes shown. After multiple rounds of 3D classification, final particle stacks were subjected to CTF refinement followed by local refinement of the masked AE1 membrane domain with imposed C2 symmetry. GS-FSC calculations indicate global resolutions of 2.98 Å (AE1-H2DIDS) and 3.07 (AE1-DEPC) applying the 0.143 cutoff. Viewing direction distribution analysis (cryoSPARC) indicates sufficient coverage. Initial models were built in PHENIX, and then further refined in ServalCat for the generation of final maps and coordinates. Calculations in cryoSPARC indicate local resolutions of lower than 3 Å around substrate and inhibitor binding sites.

Source data

Extended Data Fig. 4 Cryo-EM Structure Determination of AE1-Dipyridamole and AE1-NIF complexes.

a, b, workflow of AE1-Dipyridamole (a) and AE1-NIF (b) data processing, showing representative 2D classes, followed by 2D classification, ab initio modeling and several rounds of 3D classification, with representative 3D classes shown. After multiple rounds of 3D classification, final particle stacks were subjected to CTF refinement followed by local refinement of the masked AE1 membrane domain with imposed C2 symmetry. GS-FSC calculations indicate global resolutions of 3.13 Å (AE1-Dipyridamole) and 3.18 (AE1-NIF) applying the 0.143 cutoff. Viewing direction distribution analysis (cryoSPARC) indicates sufficient coverage. Initial models were built in PHENIX, and then further refined in ServalCat for the generation of final maps and coordinates. Calculations in cryoSPARC indicate local resolutions of lower than 3 Å around substrate and inhibitor binding sites.

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Extended Data Fig. 5 Substrate-Binding in SLC4 and SLC26 Transporters.

a, Cryo-EM density of bicarbonate and binding site residues (marine mesh) using a contour level of 6σ. b, Binding site of uracil in the UraA-Uracil structure (PDB ID: 3QE7)24. c, d, Carbonate and sodium ions in the rabbit NDCBE (SLC4A8) cryo-EM structure (PDB ID: 7RTM, EMD-24683)15, with clashes between CO32− and T538, as well as Na+ and the backbone shown as red dotted lines. Ionic interactions and hydrogen bonds are shown as grey dotted lines. e, SACP simulations allow to calculate bicarbonate affinity to AE1 anion binding site (see methods). In some structures (~5%) we have identified a second bicarbonate binding site in the putative exit channel. f, Surface display shows putative anion exit channel leading from the bicarbonate binding site to the cytoplasmic site. Channel extends below the plane of section.

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Extended Data Fig. 6 Structure and Density of AE1-Bound Inhibitors.

a, Chemical structures of different AE1 inhibitors and ligands used in this study. b, Cryo-EM density of DIDS (magenta) and key residues at a contour level of 6σ. c, H2DIDS (magenta) binding mode. d, Cryo-EM density of H2DIDS (magenta) and key residues at a contour level of 5σ. e, f, DEPC modifications of K539 and K851 (e) and corresponding cryo-EM densities (f) shown at a contour level of 8σ. g, h, Binding pocket of apo structure (g) and cryo-EM densities of residues (h) at a contour level of 6σ shown for comparison. i, Cryo-EM density of Dipyridamole (magenta) and key lysines at a contour level of 6σ. AE1’s gate and core domains are shown in tv blue and palecyan, respectively, and cryo-EM densities are shown as marine colored mesh.

Extended Data Fig. 7 Binding of NIF to AE1.

a-e, Close up of NIF binding site (a) and validation by molecular docking (b), with the best scoring docking pose shown (ΔG binding score: -52.33). c, Cryo-EM density of NIF and NIF binding site residues shown at a contour level of 3.5σ. d, Overlay of Apo (salmon) and NIF-bound AE1 (light blue) reveals subtle conformational rearrangements required for NIF binding. e, Calculation of NIF binding pocket volume and surface in POVME355. AE1’s gate and core domains are shown in tv blue and palecyan, respectively, and cryo-EM densities are shown as marine colored mesh.

Extended Data Fig. 8 Experimental testing of Compound 22 (Cmpd 22) analogs.

a, Inhibitory activity of 24 commercially available analogs of Cmpd 22 at 20 µM. Uptake experiments were performed with 2–6 technical repeats and are averaged from 3 independent experiments (n = 3). Data are represented as mean±s.e.m. b, Chemical structure of Cmpd 22 and selected analogs. c, d, Concentration response experiments show that Cmpd 22-J, Cmpd 22-R, and Cmpd 22-D have comparable inhibitory potencies as Cmpd 22 (c), while Cmpd 22-P and Cmpd 22-C appear to not inhibit AE1 (d). Concentration response uptake experiments were performed in triplicates and are averaged from 4 independent experiments (n = 4). Apparent potencies are calculated as apparent IC50 (mean) and error bars denote 95% confidence intervals. All uptake data have been normalized to Cmpd 22.

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Extended Data Table 1 Diego Antigens
Full size table
Extended Data Table 2 Calculated bicarbonate binding energies for residues in the AE1 anion binding site
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Supplementary information

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Source Data Fig. 4 and Extended Data Figs. 1–5 and 8

AE1 uptake source data.

Source Data Extended Data Fig. 1

Size-exclusion chromatogram source data, FSC curve source data and uncropped SDS–PAGE image.

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Capper, M.J., Yang, S., Stone, A.C. et al. Substrate binding and inhibition of the anion exchanger 1 transporter. Nat Struct Mol Biol 30, 1495–1504 (2023). https://doi.org/10.1038/s41594-023-01085-6

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