Sites important for Na+ and substrate binding in the Na+/proline transporter of Escherichia coli, a member of the Na+/solute symporter family.
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.
Pirch
Torsten
T
Universität Osnabrück, Fachbereich Biologie/Chemie, Abteilung Mikrobiologie, Barbarastrasse 11, D-49069 Osnabrück, Germany.
Quick
Matthias
M
Nietschke
Monika
M
Langkamp
Markus
M
Jung
Heinrich
H
D026921:Amino Acid Transport Systems, Neutral / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003545:Cysteine; D004926:Escherichia coli / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000378:metabolism; D011487:Protein Conformation; D012964:Sodium / Q000378:metabolism*; D013329:Structure-Activity Relationship
D026921:Amino Acid Transport Systems, Neutral; C033082:proline transporter; D011392:Proline; D012964:Sodium; D003545:Cysteine