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.
Keywords: amino acid transport; bioenergetics; membrane protein; membrane transport; sodium transport.
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.