Arg-52 in the Melibiose Carrier of Escherichia coli Is Important for Cation-Coupled Sugar Transport and Participates in an Intrahelical Salt Bridge

摘要

Arg-52 of the Escherichia coli melibiose carrier was replaced by Ser (R52S), Gln (R52Q), or Val (R52V). While the level of carrier in the membrane for each mutant remained similar to that for the wild type, analysis of melibiose transport showed an uncoupling of proton cotransport and a drastic reduction in Na⁺-coupled transport. Second-site revertants were selected on MacConkey plates containing melibiose, and substitutions were found at nine distinct locations in the carrier. Eight revertant substitutions were isolated from the R52S strain: Asp-19→Gly, Asp-55→Asn, Pro-60→Gln, Trp-116→Arg, Asn-244→Ser, Ser-247→Arg, Asn-248→Lys, and Ile-352→Val. Two revertants were also isolated from the R52V strain: Trp-116→Arg and Thr-338→Arg revertants. The R52Q strain yielded an Asp-55→Asn substitution and a first-site revertant, Lys-52 (R52K). The R52K strain had transport properties similar to those of the wild type. Analysis of melibiose accumulation showed that proton-driven accumulation was still defective in the second-site revertant strains, and only the Trp-116→Arg, Ser-247→Arg, and Asn-248→Lys revertants regained significant Na⁺-coupled accumulation. In general, downhill melibiose transport in the presence of Na⁺ was better in the revertant strains than in the parental mutants. Three revertant strains, Asp-19→Gly, Asp-55→Asn, and Thr-338→Arg strains, required a high Na⁺ concentration (100 mM) for maximal activity. Kinetic measurements showed that the N248K and W116R revertants lowered the Km for melibiose, while other revertants restored transport velocity. We suggest that the insertion of positive charges on membrane helices is compensating for the loss of Arg-52 and that helix II is close to helix IV and VII. We also suggest that Arg-52 is salt bridged to Asp-55 (helix II) and Asp-19 (helix I).