Trapping of inhibitor-induced conformational changes in the erythrocyte membrane anion exchanger AE1

AE1, the chloride/bicarbonate anion exchanger of the erythrocyte plasma mem brane, is highly sensitive to inhibition by stilbene disulfonate compounds such as DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonate) and DNDS (4,4'- dinitrostilbene-2,2'-disulfonate). Stilbene disulfonates recruit the anion binding site to an outward-facing conformation. We sought to identify the r egions of AE1 that undergo conformational changes upon noncovalent binding of DNDS. Since conformational changes induced by stilbene disulfonate bindi ng cause anion transport inhibition, identification of the DNDS binding reg ions may localize the substrate binding region of the protein. Cysteine res idues were introduced into 27 sites in the extracellular loop regions of an otherwise cysteineless form of AE1, called AE1C(-). The ability to label t hese residues with biotin maleimide [3-(N-maleimidylpropionyl)biocytin] was then measured in the absence and presence of DNDS, DNDS reduced the abilit y to label residues in the regions around G565, S643-M663, and S731-S742. W e interpret these regions either as (i) part of the DNDS binding site or (i i) distal to the binding site but undergoing a conformational change that s equesters the region from accessibility to biotin maleimide. DNDS alters th e conformation of residues outside the plane of the bilayer since the S643- M663 region was previously shown to be extramembranous. Upon binding DNDS, AE1 undergoes conformational changes that can be detected in extracellular loops at least 20 residues away from the hydrophobic core of the lipid bila yer. We conclude that the TM7-10 region of AE1 is central to the stilbene d isulfonate and substrate binding region of AE1.