Mechanism of melibiose/cation symport of the melibiose permease of Salmonella typhimurium

Abstract

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na+, Li+, or H+. In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na+ or Li+ and stimulated by equimolar concentrations of internal and external Na+ or Li +. Melibiose exchange is faster than efflux in the presence of H + or Na+ and stimulated by an inwardly directed Na + gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li+ stimulates efflux, and an outwardly directed Li+ gradient increases exchange, it is striking that internal and external Li+ with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2′-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na+ or Li+, exhibits appKd values of ∼1 mM for melibiose. Na + and Li+ compete for a common binding pocket with activation constants for FRET of ∼ 1mM, whereas Rb+ or Cs + exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H+ in addition to Na+ and Li +. FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2′-(N-dansyl)aminoalkyl-1-thio-β-D- galactopyranoside and Na+, Li+, or H+, which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.