Mutation of polar and charged residues in the hydrophobic NH2-terminal domains of the melibiose permease of Escherichia coli.

The suggestion that acidic residues in the hydrophobic NH2-terminal domains of Mel permease (Asp-31 in helix I, Asp-51 and Asp-55 in helix II, Asp-120 in helix IV) may be essential components of a coordination network involved in cation recognition (Pourcher, T., Zani, M.L., and Leblanc, G. (1993) J. Biol. Chem. 268, 3209-3215) is further analyzed using site-directed mutagenesis. To study whether nearby polar residues also contribute to the cation recognition process, Tyr-24, Tyr-27 and Tyr-28 (aligned with Asp-31) and Tyr-109 and Tyr-116 (aligned with Asp-120) were individually converted into a phenylalanine. The effect of replacing Arg-48 (aligned with Asp-51 and Asp-55) or Asn-83 (in the middle of helix III) by an alanine was also studied. The importance of the position of the carboxylate of the residue at position 31, 51, 55, or 120 was next examined by replacing each Asp by a Glu residue. Sugar binding and/or transport activity measurements indicate that all polar-->apolar or Asp-->Glu mutants use Na+ or Li+ for active sugar transport. Moreover, two groups of mutants could be distinguished. One group, composed of Y27F, Y28F, D31E, and Y109F mutants, retains wild type permease properties. A second group (Y24F, N83A, and Y116F and also D51E, D55E, and D120E) exhibits concomitant reduction of affinity for sodium and sugars and altered sugar specificity but conserves wild type cation selectivity profile. The data reinforce the notion that Asp-51, Asp-55, and Asp-120 residues and the position of their carboxyl side chains are of primary importance for cation recognition. Finally, since Mel permease properties are predominantly modified by mutagenizing residues located in the cytoplasmic half of the permease, we propose that Mel permease has a well-like shape opened toward the periplasmic space and is closed at its cytoplasmic extremity by a gate.