Substrate Binding and Inhibition of the Anion Exchanger 1 Transporter

Abstract

Anion Exchanger 1 (AE1, SLC4A1) is the primary bicarbonate (HCO3-) transporter expressed in erythrocyte membranes where it mediates transport of CO2 between lungs and other tissues via import/export of bicarbonate. It is also a key regulator of erythrocyte structure and antigenic recognition. Previous biochemical studies, and a low-resolution crystal structure of the transmembrane domain have provided initial insight into AE1 structure and function. However, key questions remain regarding substrate binding and transport as well as the mechanism of inhibition. The orientation of the intracellular domain as well as the localization of lipid and sterol binding sites also remain enigmatic. We herein present seven novel high resolution cryo-EM structures of the full length human transporter in the apo, bicarbonate-bound, and several inhibitor-bound states combined with uptake- and computational studies. To our knowledge, these studies represent the first full length human, and substrate bound, SLC4 transporter structure. Our results reveal important molecular details about substrate binding and transport, as well as the diverse mechanisms of AE1 inhibition by both research chemicals and prescription drugs. We also provide novel insights into the full-length transporter architecture, identify the conformational space of the Diego blood antigen system and elucidate multiple lipid and sterol binding sites.

Molecular Insights into the Effect of Cholesterol on the Binding of Bicarbonate Ions in Band 3 Protein

“…The residue R730 on TM10 of Band 3 is the most likely binding site, which agrees with the previous experiments. 6,25 Furthermore, we demonstrate that the binding stability of bicarbonate ions in Band 3 is determined by the combined interactions of several residues together with R730 imposed on the ions. CHOL in the lipid bilayer has a remarkable impact on the binding of the bicarbonate ions in Band 3, with an optimal CHOL concentration revealed for the bicarbonate ion binding.…”

“…The results shown in Figure 3 agree well with the experiments which also indicated that R730 is one of the most probable binding sites for bicarbonate ions in the Band 3 cavity. 6,25 We speculate that K539 and E535 act as bridging residues in the binding process of the bicarbonate ion, driving the anions into the Band 3 cavity and facilitating their transfer to the final binding site of R730. In the next process of the anion transport occurring in the natural plasma membrane of erythrocyte, the bicarbonate ion will separate from R730 and transport across Band 3.…”

“…Particularly, R730 is the stable binding site for bicarbonate ions, which agrees well with the experimental work. 6,25 Furthermore, we explored the effect of CHOL on the binding of bicarbonate ions in the Band 3 cavity. The proper CHOL concentration in the lipid bilayer can stabilize the conformation of the Band 3 cavity and compress the volume of the cavity, which facilitates the binding of the bicarbonate ion in the cavity.…”

Borate Transporters and SLC4 Bicarbonate Transporters Share Key Functional Properties

“…Might any of these residues interact with solute? There is no structure of a borate transporter bound to its solute, so we performed a superposition of a structure of bicarbonate-bound human SCL4A1 [ 42 ] with the AlphaFold ScBor1p model (RMSD = 3.149 Å) ( Figure 4 F) [ 29 ]. As suspected, the bicarbonate places in the cavity formed where TM3 and TM10 meet.…”

“…Among amino acids identified in this study, Q396 and Y212 are closest to where bicarbonate superposes. In human SLC4A1, R730 is homologous with Q396 and is observed to interact directly with bicarbonate [ 42 ]. Interestingly, the carboxylate group of D347 is located 8.2 Å away from the superposed bicarbonate carbon.…”