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Crystal structures of transporters with a LeuT-type structural fold assign core transmembrane domain 6 (TM6') a central role in substrate binding and translocation. Here, the function of TM6' in the sodium/proline symporter PutP, a member of the solute/sodium symporter family, was investigated. A complete scan of TM6' identified eight amino acids as particularly important for PutP function. Of these residues, Tyr-248, His-253, and Arg-257 impact sodium binding, whereas Arg-257 and Ala-260 may participate in interactions leading to closure of the inner gate. Furthermore, the previous suggestion of an involvement of Trp-244, Tyr-248, and Pro-252 in proline binding is further supported. In addition, substitution of Gly-245, Gly-247, and Gly-250 affects the amount of PutP in the membrane. A Cys accessibility analysis suggests an involvement of the inner half of TM6' in the formation of a hydrophilic pathway that is open to the inside in the absence of ligands and closed in the presence of sodium and proline. In conclusion, the results demonstrate that TM6' plays a central role in substrate binding and release on the inner side of the membrane also in PutP and extend the knowledge on functionally relevant amino acids in transporters with a LeuT-type structural fold., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Na(+)/solute symporters are essential membrane integrated proteins that couple the flow of Na(+) ions driven by electrochemical Na(+) gradients to the transport of solutes across biological membranes. Here, we used a combination of molecular modeling techniques and evolutionary conservation analysis to construct and validate a first model of the Na(+)/proline symporter PutP of Escherichia coli based on the crystal structure of the bacterial Na(+)/galactose symporter vSGLT. Ligand docking experiments were employed to gain information about residues involved in proline binding. The proposed model is consistent with the available experimental data and was further validated by amino acid substitutions and kinetic and protein chemical analyses. Combination of the results of molecular modeling and functional studies predicts the location and organization of the Na(+) and proline binding sites. Remarkably, as proposed computationally and discovered here experimentally, residues Y140, W244, and Y248 of transmembrane segments 4 and 7 are found to be particularly important for PutP function and suggested to participate in proline binding and/or gating., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Abstract: Na+/solute symporters are essential membrane integrated proteins that couple the flow of Na+ ions driven by electrochemical Na+ gradients to the transport of solutes across biological membranes. Here, we used a combination of molecular modeling techniques and evolutionary conservation analysis to construct and validate a first model of the Na+/proline symporter PutP of Escherichia coli based on the crystal structure of the bacterial Na+/galactose symporter vSGLT. Ligand docking experiments were employed to gain information about residues involved in proline binding. The proposed model is consistent with the available experimental data and was further validated by amino acid substitutions and kinetic and protein chemical analyses. Combination of the results of molecular modeling and functional studies predicts the location and organization of the Na+ and proline binding sites. Remarkably, as proposed computationally and discovered here experimentally, residues Y140, W244, and Y248 of transmembrane segments 4 and 7 are found to be particularly important for PutP function and suggested to participate in proline binding and/or gating. [Copyright &y& Elsevier]