Format

Send to

Choose Destination
J Biol Chem. 2014 Jan 17;289(3):1377-87. doi: 10.1074/jbc.M113.510743. Epub 2013 Nov 20.

A L-lysine transporter of high stereoselectivity of the amino acid-polyamine-organocation (APC) superfamily: production, functional characterization, and structure modeling.

Author information

1
From the Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main 60438, Germany.

Abstract

Membrane proteins of the amino acid-polyamine-organocation (APC) superfamily transport amino acids and amines across membranes and play an important role in the regulation of cellular processes. We report the heterologous production of the LysP-related transporter STM2200 from Salmonella typhimurium in Escherichia coli, its purification, and functional characterization. STM2200 is assumed to be a proton-dependent APC transporter of L-lysine. The functional interaction between basic amino acids and STM2200 was investigated by thermoanalytical methods, i.e. differential scanning and isothermal titration calorimetry. Binding of L-lysine to STM2200 in its solubilized monomer form is entropy-driven. It is characterized by a dissociation constant of 40 μm at pH 5.9 and is highly selective; no evidence was found for the binding of L-arginine, L-ornithine, L-2,4-diaminobutyric acid, and L-alanine. D-lysine is bound 45 times more weakly than its L-chiral form. We thus postulate that STM2200 functions as a specific transport protein. Based on the crystal structure of ApcT (Shaffer, P. L., Goehring, A., Shankaranarayanan, A., and Gouaux, E. (2009) Science 325, 1010-1014), a proton-dependent amino acid transporter of the APC superfamily, a homology model of STM2200 was created. Docking studies allowed identification of possible ligand binding sites. The resulting predictions indicated that Glu-222 and Arg-395 of STM2200 are markedly involved in ligand binding, whereas Lys-163 is suggested to be of structural and functional relevance. Selected variants of STM2200 where these three amino acid residues were substituted using single site-directed mutagenesis showed no evidence for L-lysine binding by isothermal titration calorimetry, which confirmed the predictions. Molecular aspects of the observed ligand specificity are discussed.

KEYWORDS:

APC Superfamily; Amino Acid Transport; Homology Modeling; Isothermal Titration Calorimetry; Protein Purification; Quantitative l-Lysine Binding; Secondary Active Transporter; Site-directed Mutagenesis

PMID:
24257746
PMCID:
PMC3894322
DOI:
10.1074/jbc.M113.510743
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center