Mutagenesis of acidic residues in putative membrane-spanning segments of the melibiose permease of Escherichia coli. I. Effect on Na(+)-dependent transport and binding properties.

Four aspartic acids, distributed in different putative membrane-spanning segments of the NH2-terminal domain of melibiose (mel) permease (D31 in helix I, D51 and D55 in helix II, and D120 in helix IV) were individually replaced by either Asn or Cys using site-directed mutagenesis. mel permease with either neutral residues at position 51, 55, or 120 or permease with a Cys in place of D31 does not catalyze significant Na(+)-linked methyl-1-thio-beta-D-galactopyranoside (TMG) accumulation. Binding studies of a high affinity ligand (p-nitrophenyl-alpha-D-galactopyranoside (NPG)) on de-energized membrane vesicles indicate that these modified transporters (i) retain the ability to bind the alpha-galactosides NPG or melibiose and the beta-galactoside TMG and (ii) exhibit a Na(+)-independent sugar-binding phenotype. In contrast, mel permease with an Asn residue at position 31 mediates Na(+)-coupled TMG transport and displays a Na(+)-dependent sugar binding phenotype, but requires a higher concentration of sodium than wild-type permease to produce maximal stimulation of sugar binding. The observation that individual mutation of the Asp residue at position 31, 51, 55, or 120 systematically and selectively modifies the contribution of the coupling ion to the early step of the transport reaction, i.e. cosubstrate binding, raises the possibility that (i) these 4 aspartic residues are at or near the cationic binding site of mel permease, (ii) the NH2-terminal domain of mel permease in which they are distributed accommodates or is part of the cationic binding site, and (iii) the oxygen atoms of these Asp side chains contribute to coordination of the coupling ion.