Effects of mutations and ligands on the thermostability of the L-arginine/agmatine antiporter adic and deduced insights into ligand-binding of human L-type amino acid transporters

The L-arginine/agmatine transporter AdiC is a prokaryotic member of the SLC7 family, which enables pathogenic enterobacteria to survive the extremely acidic gastric environment. Wild-type AdiC from Escherichia coli, as well as its previously reported point mutants N22A and S26A, were overexpressed homologously and purified to homogeneity. A size-exclusion chromatography-based thermostability assay was used to determine the melting temperatures (T_m s) of the purified AdiC variants in the absence and presence of the selected ligands L-arginine (Arg), agmatine, L-arginine methyl ester, and L-arginine amide. The resulting T_m s indicated stabilization of AdiC variants upon ligand binding, in which T_m s and ligand binding affinities correlated positively. Considering results from this and previous studies, we revisited the role of AdiC residue S26 in Arg binding and proposed interactions of the α-carboxylate group of Arg exclusively with amide groups of the AdiC backbone. In the context of substrate binding in the human SLC7 family member L-type amino acid transporter-1 (LAT1; SLC7A5), an analogous role of S66 in LAT1 to S26 in AdiC is discussed based on homology modeling and amino acid sequence analysis. Finally, we propose a binding mechanism for L-amino acid substrates to LATs from the SLC7 family.