title: Core Transmembrane Domain 6 Plays a Pivotal Role in the Transport Cycle of the Sodium/Proline Symporter PutP.
abstract: 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.
authors: From the Division of Microbiology, Department of Biology 1, Ludwig Maximilians University Munich, D-82152 Martinsried, Germany.
authors: Susanne
authors: S
authors: Bracher
authors: From the Division of Microbiology, Department of Biology 1, Ludwig Maximilians University Munich, D-82152 Martinsried, Germany.
authors: Claudia C
authors: CC
authors: Schmidt
authors: From the Division of Microbiology, Department of Biology 1, Ludwig Maximilians University Munich, D-82152 Martinsried, Germany.
authors: Sophie I
authors: SI
authors: Dittmer
authors: From the Division of Microbiology, Department of Biology 1, Ludwig Maximilians University Munich, D-82152 Martinsried, Germany hjung@lmu.de.
authors: Heinrich
authors: H
authors: Jung
mesh_terms: D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017136:Ion Transport / Q000502:physiology; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D017510:Protein Folding; D012964:Sodium / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D027981:Symporters / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D026921:Amino Acid Transport Systems, Neutral; D029968:Escherichia coli Proteins; D027981:Symporters; C060609:PutP protein, E coli; D011392:Proline; D012964:Sodium
keywords: amino acid transport; bioenergetics; membrane protein; membrane transport; sodium transport