PMID:11756453
| Citation |
Pirch, T, Quick, M, Nietschke, M, Langkamp, M and Jung, H (2002) Sites important for Na+ and substrate binding in the Na+/proline transporter of Escherichia coli, a member of the Na+/solute symporter family. J. Biol. Chem. 277:8790-6 |
|---|---|
| Abstract |
To elucidate the functional importance of transmembrane domain II 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. |
| Links |
PubMed Online version:10.1074/jbc.M111008200 |
| Keywords |
Amino Acid Transport Systems, Neutral/chemistry; Amino Acid Transport Systems, Neutral/metabolism; Binding Sites; Cysteine; Escherichia coli/metabolism; Mutagenesis, Site-Directed; Proline/metabolism; Protein Conformation; Sodium/metabolism; Structure-Activity Relationship |
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