Arg-52 in the melibiose carrier of Escherichia coli is important for cation-coupled sugar transport and participates in an intrahelical salt bridge.

Bacterial secondary active transporters capture free energy from the movement of cations down their electrochemical gradient and use it to drive the transport of solutes such as sugars, amino acids, Krebs cycle intermediates, antibiotics, and inorganic ions across the cell membrane (27, 31, 33). The melibiose carrier (MelB) of Escherichia coli is a cation/substrate symporter which couples transport of Na+ and melibiose across the bacterial inner membrane (for reviews see references 22, 32, and 38). In addition to melibiose, MelB transports a variety of sugar substrates including α- and β-galactosides as well as some monosaccharides (46, 50). An interesting feature of MelB is its ability to couple sugar transport to three different cations, Na+, Li+, and H+, depending on the configuration of the transported sugar (44–46, 50). Sugar binding studies using membrane vesicles have shown that the presence of Na+ and Li+ ions increases the carrier’s affinity for galactosides and that the cations compete for a single binding site (6, 8). The melB gene has been cloned (18) and sequenced (52). The primary amino acid sequence deduced from the gene sequence predicts a hydrophobic protein (70% apolar) with a molecular mass of 52 kDa (52). The results of hydropathy analysis and melB-phoA fusions have provided good evidence for a two-dimensional structure where the protein forms 12 α-helical transmembrane domains connected by hydrophilic loops (1, 34, 52). E. coli MelB is a member of the galactoside-pentose-hexuronide family of bacterial transport proteins (32). The MelB subfamily consists of melibiose carriers from E. coli, Salmonella typhimurium, Klebsiella pneumoniae, and Enterobacter aerogenes. Although a high degree (78 to 85%) of amino acid identity exists among carriers in the MelB subfamily (30, 32), there are distinct differences in cation selectivity. For example, the MelB of E. coli couples H+, Na+, and Li+ to sugar transport, while the K. pneumoniae carrier couples either H+ or Li+, but not Na+ (16). The amino acid residues responsible for Na+ recognition were localized by constructing chimeras of the E. coli and K. pneumoniae melibiose carriers (15). Replacement of the first 81 amino acids of the K. pneumoniae carrier with those of the E. coli MelB was sufficient to allow the K. pneumoniae carrier to couple Na+ and sugar transport (15). Interestingly, a single-amino-acid substitution in helix II of K. pneumoniae, Ala-58→Asn, also resulted in Na+-coupled sugar transport (17). Cation recognition in E. coli MelB has also been investigated by site-directed mutagenesis. Studies have focused primarily on acidic residues that reside on membrane-spanning helices in the amino-terminal portion of the carrier. Neutral amino acid substitutions for Asp-19 (helix I), Asp-55 (helix II), Asp-59 (helix II), and Asp-124 (helix IV) cause the loss of Na+-coupled sugar transport (32, 35, 36, 53). In these mutants, sugar binding is comparable to that of wild-type MelB in the absence of Na+, but this binding is no longer stimulated by Na+. Taken together, the results of these studies have led to a model in which Asp residues at positions 19, 55, 59, and 124 provide part of a network for the coordination of cations in E. coli MelB (22, 32, 36, 54). The studies mentioned above show that the acidic amino acids on transmembrane helix II of the E. coli MelB, Asp-tt and Asp-59, are important for cation recognition. While the roles of these aspartates have been studied thoroughly, less is known about the positively charged residue Arg-52 in this helix. It has been reported that a substitution of Ala for Arg-52 leads to a 95% loss in carrier activity but that the remaining activity is still stimulated by Na+ and Li+ (54). In the present study, we further investigate the role of Arg-52. We use site-directed mutagenesis to substitute Gln, Val, and Ser for Arg-52 (R52Q, R52V, and R52S, respectively). We show that substitution of Arg-52 causes a dramatic loss of melibiose transport, with only a small amount of Na+-stimulated activity remaining. Subsequently, we use the Val-52, Ser-52, and Gln-52 mutant strains to isolate revertant strains which regain the ability to transport melibiose. Sequence analyses reveal that revertant mutations, with one exception, are found at locations other than position 52. The majority of these mutations result in substitution of amino acids located on transmembrane domains in both the amino and carboxyl halves of the protein. Our analysis of the melibiose transport properties in the strains with site-directed or second-site revertant mutations provide significant new information about the functional role of Arg-52. On the basis of our data, we suggest that specific transmembrane helices are close to one another in the three-dimensional structure of the protein. The data also suggest that Arg-52 is involved in an intrahelical salt bridge with Asp-55 and possibly in an interhelical salt bridge with Asp-19.