title: 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 (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, 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.
authors.affiliation: Department of Cell Physiology and Molecular Biophysics, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA. Lan.Guan@ttuhsc.edu
authors.forename: Lan
authors.initials: L
authors.lastname: Guan
authors.forename: Shailika
authors.initials: S
authors.lastname: Nurva
authors.forename: Siva P
authors.initials: SP
authors.lastname: Ankeshwarapu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D008553:Melibiose / Q000235:genetics / Q000378:metabolism*; D008670:Metals / Q000378:metabolism; D008954:Models, Biological; D020836:Protein Structure, Quaternary; D011522:Protons; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism*; D027981:Symporters
chemical_list: D001426:Bacterial Proteins; D008670:Metals; D011522:Protons; D027981:Symporters; C021548:melibiose permease; D008553:Melibiose; D002118:Calcium