Evidence for a close association between helix IV and helix XI in the melibiose carrier of Escherichia coli

AbstractThe melibiose carrier of Escherichia coli is a cation–sugar cotransport protein. Asp124 in membrane-spanning helix IV of the carrier protein was replaced with Ser, Ile or Phe by site-directed mutagenesis of a plasmid containing the melB gene. Each of these mutants failed to show membrane transport of melibiose and melibiose-positive revertants could be isolated on melibiose MacConkey indicator plates. D124F showed only one type of revertant (D124C) and D124I showed only revertants to the normal (D124). Second site revertants were not found with either of these mutants. S124, however, showed two types of second site revertants: D124S/V375A and D124S/V375G. The revertant D124S/V375A had lost melibiose/proton cotransport but showed 25% of normal melibiose (20 mM) uptake in the presence of 10 mM sodium ion at 37°C. The value for the parental strain D124S was 2%. The second revertant D124S/V375G showed greater activity than S124 in the presence of 100 mM NaCl at both 20°C and 37°C. It was concluded that in the normal carrier protein Asp124 in helix IV is probably close to Val375 in helix XI. Since Lys377 is close to Val375, it is possible that Asp124 may interact with Lys377 to form a salt bridge