Sites important for Na+ and substrate binding in the Na+/proline transporter of Escherichia coli, a member of the Na+/solute symporter family

Autor(en): Pirch, T
Quick, M
Nietschke, M
Langkamp, M
Jung, H
Stichwörter: ACIDIC RESIDUES; Biochemistry & Molecular Biology; EXPRESSION; MELIBIOSE PERMEASE; MEMBRANE-SPANNING SEGMENTS; NA+/GLUCOSE COTRANSPORTER; PROLINE UPTAKE; PROTEIN; PURIFICATION; RECONSTITUTION; TOPOLOGY
Erscheinungsdatum: 2002
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 277
Ausgabe: 11
Startseite: 8790
Seitenende: 8796
Zusammenfassung: 
To elucidate the functional importance of transmembrane domain 11 in the Na+/proline transporter (PutP) of Escherichia coli we analyzed the effect of replacing Ser-54 through Gly-58. Substitution of Asp-55 or Met-56 dramatically reduces the apparent affinity for Na+ and Li+ in a cation-dependent manner. Conversely, Cys in place of Gly-58 significantly reduces only the apparent proline affinity while substitution of Ser-57 results in a dramatic reduction of the apparent proline and cation affinities. Interestingly, upon increasing the proline concentration the apparent Na+ affinity of Ser-57 replacement mutants converges toward the wild-type value, indicating a close cooperativity between cation and substrate site(s). This notion is supported by the fact that Na+-stimulated site-specific fluorescence labeling of a single Cys at position 57 is completely reversed by the addition of proline. Similar results are obtained upon labeling of a Cys at position 54 or 58. Taken together, these results indicate that Asp-55 and Met-56 are located at or close to the ion-binding site while Ser-54, Ser-57, and Gly-58 may be close to the proline translocation pathway. In addition, the data prod at an involvement of the latter residues in ligand-induced conformational dynamics that are crucial for cation-coupled transport.
ISSN: 00219258
DOI: 10.1074/jbc.M111008200

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