Substrate binding and inhibition of the anion exchanger 1 transporter

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. Access through your institution Buy or subscribe This is a preview of subscription content, access via

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subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL AND FUNCTIONAL INSIGHTS INTO THE LIPID REGULATION OF HUMAN ANION EXCHANGER 2

Article Open access 26 January 2024 STRUCTURAL INSIGHTS INTO HUMAN ORGANIC CATION TRANSPORTER 1 TRANSPORT AND INHIBITION Article Open access 15 March 2024 MECHANISM OF ANION EXCHANGE AND

SMALL-MOLECULE INHIBITION OF PENDRIN Article Open access 06 January 2024 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. REFERENCES * Elgsaeter, A., Stokke,

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references 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. AUTHOR INFORMATION Author notes * These authors

contributed equally: Michael J. Capper, Shifan Yang, Alexander C. Stone. AUTHORS AND AFFILIATIONS * 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 * 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 * 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 * Department of

Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY, USA Gregory Zilberg, Yamuna Kalyani Mathiharan, Bin Zhang & Daniel Wacker * Department of Artificial Intelligence and

Human Health, Icahn School of Medicine at Mount Sinai, New York, NY, USA Avner Schlessinger & Bin Zhang * Icahn Genomics Institute, Icahn School of Medicine at Mount Sinai, New York,

NY, USA Bin Zhang Authors * Michael J. Capper View author publications You can also search for this author inPubMed Google Scholar * Shifan Yang View author publications You can also search

for this author inPubMed Google Scholar * Alexander C. Stone View author publications You can also search for this author inPubMed Google Scholar * Sezen Vatansever View author publications

You can also search for this author inPubMed Google Scholar * Gregory Zilberg View author publications You can also search for this author inPubMed Google Scholar * Yamuna Kalyani Mathiharan

View author publications You can also search for this author inPubMed Google Scholar * Raul Habib View author publications You can also search for this author inPubMed Google Scholar *

Keino Hutchinson View author publications You can also search for this author inPubMed Google Scholar * Yihan Zhao View author publications You can also search for this author inPubMed 

Google Scholar * Avner Schlessinger View author publications You can also search for this author inPubMed Google Scholar * Mihaly Mezei View author publications You can also search for this

author inPubMed Google Scholar * Roman Osman View author publications You can also search for this author inPubMed Google Scholar * Bin Zhang View author publications You can also search for

this author inPubMed Google Scholar * Daniel Wacker View author publications You can also search for this author inPubMed Google Scholar 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. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

interests. PEER REVIEW PEER REVIEW INFORMATION _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. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional

affiliations. 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. Source data 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. Source data 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. Source data SUPPLEMENTARY INFORMATION REPORTING SUMMARY PEER REVIEW FILE SOURCE DATA 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

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 14 December 2022 * Accepted: 28 July 2023 * Published: 07 September 2023 * Issue Date: October

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