Abstract
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 (Tms) 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 Tms indicated stabilization of AdiC variants upon ligand binding, in which Tms 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.
Highlights
Extreme acid resistance systems enable pathogenic enterobacteria such as Escherichia coli strain O157:H7 to survive the strongly acidic environment of the stomach and subsequently colonize and infect the human gut
To explore the thermostabilities of AdiC variants in the absence and presence of selected ligands (200-fold molar excess), His-tagged AdiC-wt, AdiC-N22A, and AdiC-S26A proteins were overexpressed in E. coli, purified by nickel affinity chromatography, and depleted of their His-tag by human rhinovirus 3C protease treatment
Keeping in mind the results from thermostability, one might speculate that purified AdiC-N22A is present in the outward open conformation and that upon addition of Arg binding of this substrate induces a conformational change of the transporter into the outward-facing occluded state, which significantly stabilizes the complex (∆Tm = 8.2 ◦C) and traps the ligand
Summary
Extreme acid resistance systems enable pathogenic enterobacteria such as Escherichia coli strain O157:H7 to survive the strongly acidic environment of the stomach and subsequently colonize and infect the human gut One of these systems is the arginine-dependent resistance system of E. coli, which consists of the decarboxylase AdiA and the antiporter AdiC [1]. Six crystal structures of AdiC have been reported so far, which were elucidated in different states, and in the absence and presence of substrate, i.e., in the outward-open, substrate-free [4,5,6], outward-open, L-arginine- [7] and agmatine-bound [6], and outward-facing occluded L-arginine-bound states [8] These three-dimensional (3D) structures are of great value for the understanding of the molecular working mechanism of AdiC, and of other APC superfamily transporters including mammalian SLC7 family members. Homology modeling combined with structure-function studies or structure-based ligand discovery were reported for human and mammalian SLC7 family members, e.g., for the L-amino acid transporter-1 (LAT1; SLC7A5) [9,10,11,12,13,14,15,16] and -2 (LAT2; SLC7A8) [17,18,19,20]
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