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

Abstract

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 chromatographybased 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.

Molecular mechanism of substrate selectivity of the arginine-agmatine Antiporter AdiC

“…4 ). It has been proposed that Ser26 does not govern ligand selectivity through sidechain H bond interactions, since a fully transport-active AdiC S26A mutant shows a normal capacity to distinguish Arg + from Arg +2 11 , 30 . However experimental and theoretical studies, however, have shown that water molecules can, in some cases, take over the role of the mutated residue within the binding pocket 31 , 32 .…”

L amino acid transporter structure and molecular bases for the asymmetry of substrate interaction

“…All of these bacterial transporters belong to the APC family, within the APC superfamily, and adopt the APC superfamily-fold, originally described for the bacterial transporter LeuT 24 . Structural models based on AdiC and ApcT have been used to obtain clues regarding substrate recognition by LAT1 25 and LAT2 16,26 . However, due to the low sequence identity with human LATs (14–22%) these models lack the robustness required to dissect the molecular mechanisms underlying transport, study the molecular defects associated with human mutations, and build structure-guided inhibitors with potential pharmacological applications.…”

In silico Description of LAT1 Transport Mechanism at an Atomistic Level

“…Despite its biochemical and pharmacological interest, LAT1 structure has not been solved yet by crystallography. Some LAT1 comparative models have already been proposed, mostly based on the crystallographic structure of AdiC in the OF conformation (Dickens et al, 2013 ; Geier et al, 2013 ; Augustyn et al, 2016 ; Colas et al, 2016 ; Napolitano et al, 2017a ; Ilgü et al, 2018 ) which covers both the open and closed LAT1 conformations. However, since these models are all based on the only available OF conformation of different AdiC crystallographic structures, it is currently unknown which residues are actually involved in the substrate transport when LAT1 is in its IF conformation.…”