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  A L-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily PRODUCTION, FUNCTIONAL CHARACTERIZATION, AND STRUCTURE MODELING

Kaur, J., Olkhova, E., Malviya, V. N., Grell, E., & Michel, H. (2014). A L-Lysine Transporter of High Stereoselectivity of the Amino Acid-Polyamine-Organocation (APC) Superfamily PRODUCTION, FUNCTIONAL CHARACTERIZATION, AND STRUCTURE MODELING. The Journal of Biological Chemistry, 289(3), 1377-1387. doi:10.1074/jbc.M113.510743.

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 Creators:
Kaur, Jagdeep1, Author           
Olkhova, Elena1, Author           
Malviya, Viveka Nand1, Author           
Grell, Ernst1, Author           
Michel, Hartmut1, Author                 
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1Department of Molecular Membrane Biology, Max Planck Institute of Biophysics, Max Planck Society, ou_2068290              

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 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 40μM 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.

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Language(s): eng - English
 Dates: 201420142014-01-17
 Publication Status: Published in print
 Pages: 12
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1074/jbc.M113.510743
 Degree: -

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Title: The Journal of Biological Chemistry
  Other : JBC
Source Genre: Journal
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Publ. Info: Baltimore, etc. : American Society for Biochemistry and Molecular Biology [etc.]
Pages: 12 Volume / Issue: 289 (3) Sequence Number: - Start / End Page: 1377 - 1387 Identifier: ISSN: 0021-9258
CoNE: https://pure.mpg.de/cone/journals/resource/954925410826_1