title: A L-lysine transporter of high stereoselectivity of the amino acid-polyamine-organocation (APC) superfamily: production, functional characterization, and structure modeling.
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.
authors.affiliation: From the Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Frankfurt am Main 60438, Germany.
authors.forename: Jagdeep
authors.initials: J
authors.lastname: Kaur
authors.forename: Elena
authors.initials: E
authors.lastname: Olkhova
authors.forename: Viveka Nand
authors.initials: VN
authors.lastname: Malviya
authors.forename: Ernst
authors.initials: E
authors.lastname: Grell
authors.forename: Hartmut
authors.initials: H
authors.lastname: Michel
mesh_terms: D019943:Amino Acid Substitution; D026923:Amino Acid Transport Systems, Basic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D062105:Molecular Docking Simulation; D020125:Mutation, Missense; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012486:Salmonella typhimurium / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D040681:Structural Homology, Protein; D013329:Structure-Activity Relationship
chemical_list: D026923:Amino Acid Transport Systems, Basic; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D008239:Lysine
keywords: APC Superfamily; Amino Acid Transport; Homology Modeling; Isothermal Titration Calorimetry; Protein Purification; Quantitative l-Lysine Binding; Secondary Active Transporter; Site-directed Mutagenesis