Conformational transition induced in the aspartate:alanine antiporter by L-Ala binding

An aspartate:alanine antiporter (AspT) from the lactic acid bacterium Tetragenococcus halophilus catalyzes the electrogenic aspartate^1-:alanine⁰ exchange reaction. Our previous kinetic analyses of transport reactions mediated by AspT in reconstituted liposomes suggested that, although the substrate transport reactions are physiologically coupled, the putative binding sites of L-aspartate (-Asp) and L-alanine (-Ala) are independently located on AspT. By using the fluorescent probe Oregon Green maleimide (OGM), which reacts specifically with cysteine, we also found that the presence of L-Asp changes the conformation of AspT. In this study, we conducted an OGM labeling assay in the presence of L-Ala. The labeling efficiency of single cysteine mutants (G62C and P79C) in transmembrane helix 3 of the AspT showed novel patterns depending on the presence of L-Ala or analogs. A concentration-dependent shift of AspT from the conformation in the presence of one substrate to that specific to the substrate added subsequently (L-Ala or L-Asp) was observed. Moreover, size-exclusion-chromatography-based thermostability assays indicated that the thermal stability of AspT in the presence of L-Ala differed from that in the presence of L-Asp. From these results, we concluded that L-Ala binding yields a conformation different from the apo or L-Asp binding conformations.