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. 2018 Mar 20;19(3):918.
doi: 10.3390/ijms19030918.

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

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Free PMC article

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

Hüseyin Ilgü et al. Int J Mol Sci. .
Free PMC article

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.

Keywords: ">l-arginine/agmatine transporter; ">l-type amino acid transporter; AdiC; LAT1; acid resistance; cancer metabolism; enterobacteria; melting temperature; thermostability.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
View into the substrate binding sites of the AdiC crystal structures in the outward-open, substrate-free (apoAdiC-wt; PDB ID code: 5J4I) (A); and outward-facing, occluded, l-arginine-bound states (ArgAdiC-N22A; PDB ID code: 3L1L) (B). Amino acid side chains located in the substrate binding pockets and reported to interact with substrates are displayed as sticks. The exception represents N22, which is located near the substrate binding site and was not reported to interact with substrates in the currently available crystal structures. The volumes of the substrate binding pockets (indicated and colored in dark orange) are different in the outward-open and outward-facing occluded states because of the different protein conformations and positions of residue W202. Residues N22 and S26, which are pertinent to the presented study, are colored in magenta and black in the apoAdiC-wt and ArgAdiC-N22A structures, respectively. The bound Arg molecule in the ArgAdiC-N22A structure is colored in yellow. The apoAdiC-wt and ArgAdiC-N22A structures are shown as ribbons colored in light-yellow and light-magenta. Besides N22 and S26, amino acid side chains involved in substrate binding are colored in salmon and light-blue in the apoAdiC-wt and ArgAdiC-N22A structures, respectively. The hydrogen bond between S26 and the α-carboxylate group of Arg is indicated by a dotted line, as well as the distance in Å.
Figure 2
Figure 2
Workflow for Tm determination of ligand-free and -bound membrane protein using SEC. Purified membrane protein samples in the absence and presence of selected ligands are exposed to different temperatures for a defined time period and then subjected to SEC using a thermocycler and an FPLC, respectively. Peak heights in elution profiles enable the quantification of the fraction of membrane protein that remains intact after heat treatment. Plotting of remaining fractions versus temperatures results in melting curves from which Tm values are determined. Comparison of Tms allows determination of possible ligand-induced stabilization effects on purified membrane proteins, e.g., right shift of the blue melting curve (protein with ligand), indicating increased Tm compared to the red curve (protein without ligand). The flow chart was adapted from Mancusso et al. (2011) [24]. Membrane protein structures without and with bound ligand are coloured in red and blue (ligand in magenta), respectively.
Figure 3
Figure 3
SDS-PAGE analysis of purified AdiC variants. A Coomassie brilliant-blue stained 13.5% SDS/polyacrylamide gel of wild-type AdiC (wt), and N22A and S26A AdiC mutants (5 µg of protein loaded per lane) is shown.
Figure 4
Figure 4
Thermostability curves and resulting melting temperatures of AdiC-wt, AdiC-N22A, and AdiC-S26A in the absence and presence of selected ligands. Ligands: l-arginine (Arg), agmatine (Agm), l-arginine methyl ester (Arg-OMe), and l-arginine amide (Arg-NH2). Tm: melting temperature. The determined Tm values are from at least three independent experiments, each in triplicate, and 95% confidence interval values are indicated below Tms. Error bars represent SEM.
Figure 5
Figure 5
Amino acid sequence comparison between AdiC from E. coli and human l-amino acid transporters (LATs) from the SLC7 family. Identity and similarity values between different sequences are indicated in percentages and color scored. The Asc-2 (Slc7a12) and arpAT (Slc7a15) LATs were not considered in the sequence analysis, because their genes are not present or highly inactivated in primate genomes [26].
Figure 6
Figure 6
Homology model of human LAT1 with docked l-Phe substrate (PheLAT1) and l-amino acid binding hypothesis for LATs. (A) View into the substrate binding site (colored in dark orange) of the AdiC structure-based human LAT1 homology model [9]. Oxygen and nitrogen atoms from carbonyl and amide groups of protein backbone amino acid residues (displayed as sticks and colored in grey) that are in hydrogen bond distance to the α-amino and α-carboxyl groups of the substrate l-Phe (colored in gold) are indicated as dotted lines. Interatomic distances are indicated, and numbers correspond to Å. The PheLAT1 model is shown as ribbon colored in light-grey. (B) Potential binding mechanism of l-amino acid substrates to LATs from the SLC7 family. Based on the LAT1 homology model (A) [9] and the available AdiC structures with bound substrates [6,8], protein backbone interactions via carbonyl and amide groups with the α-amino and α-carboxyl group of the amino acid substrates are proposed. For interactions with the variable R-group of amino acid substrates, one backbone interaction and arbitrary amino acid side chains are displayed.

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References

    1. Foster J.W. Escherichia coli acid resistance: Tales of an amateur acidophile. Nat. Rev. Microbiol. 2004;2:898–907. doi: 10.1038/nrmicro1021. - DOI - PubMed
    1. Casagrande F., Ratera M., Schenk A.D., Chami M., Valencia E., Lopez J.M., Torrents D., Engel A., Palacin M., Fotiadis D. Projection structure of a member of the amino acid/polyamine/organocation transporter superfamily. J. Biol. Chem. 2008;283:33240–33248. doi: 10.1074/jbc.M806917200. - DOI - PMC - PubMed
    1. Fotiadis D., Kanai Y., Palacin M. The SLC3 and SLC7 families of amino acid transporters. Mol. Asp. Med. 2013;34:139–158. doi: 10.1016/j.mam.2012.10.007. - DOI - PubMed
    1. Gao X., Lu F., Zhou L., Dang S., Sun L., Li X., Wang J., Shi Y. Structure and mechanism of an amino acid antiporter. Science. 2009;324:1565–1568. doi: 10.1126/science.1173654. - DOI - PubMed
    1. Fang Y., Jayaram H., Shane T., Kolmakova-Partensky L., Wu F., Williams C., Xiong Y., Miller C. Structure of a prokaryotic virtual proton pump at 3.2 Å resolution. Nature. 2009;460:1040–1043. doi: 10.1038/nature08201. - DOI - PMC - PubMed

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