Role of Gly117 in the Cation/Melibiose Symport of MelB of Salmonella typhimurium

Abstract

The melibiose permease of Salmonella typhimurium (MelB St ) catalyzes symport of melibiose with Na + , Li + , or H + , and bioinformatics analysis indicates that a conserved Gly117 (helix IV) is part of the Na + -binding site. We mutated Gly117 to Ala, Pro, Trp, or Arg; the effects on melibiose transport and binding of cosubstrates depended on the physical−chemical properties of the side chain. Compared with WT MelB St , the Gly117 → Ala mutant exhibited little difference in either cosubstrate binding or stimulation of melibiose transport by Na + or Li + , but all other mutations reduced melibiose active transport and efflux, and decreased the apparent affinity for Na + . The bulky Trp at position 117 caused the greatest inhibition of melibiose binding, and Gly117 → Arg yielded less than a 4-fold decrease in the apparent affinity for melibiose at saturating Na + or Li + concentration. Remarkably, the mutant Gly117 → Arg catalyzed melibiose exchange in the presence of Na + or Li + , but did not catalyze melibiose translocation involving net flux of the coupling cation, indicating that sugar is released prior to release of the coupling cation. Taken together, the findings are consistent with the notion that Gly117 plays an important role in cation binding and translocation.

Reduced Na + Affinity Increases Turnover of Salmonella enterica Serovar Typhimurium MelB

“…The 5-h cultures with the expressed MelB St were washed with 20 mM Tris-HCl (pH 7.5). The preparation of crude membranes was carried out as described previously (15). After a protein assay using a Microbicinchoninic acid protein assay kit (Pierce), 25 g of crude membranes was loaded onto each well of an SDS-12% PAGE plate.…”

“…he melibiose permease of Salmonella enterica serovar Typhimurium (MelB St ) shares 86% identity and 96% similarity to the primary sequence of its Escherichia coli orthologue (MelB Ec ) (45,15). Like MelB Ec (1, 8, 12-14, 23-25, 27, 29, 35), MeB St catalyzes symport of galactoside with Na ϩ , Li ϩ , or H ϩ (15,18), utilizing the free energy from the downhill translocation of one cosubstrate to drive uphill translocation of the other (3,39,40,42,44), and all three cations compete for a common binding pocket (6,18,26,31).…”

“…Like MelB Ec (1, 8, 12-14, 23-25, 27, 29, 35), MeB St catalyzes symport of galactoside with Na ϩ , Li ϩ , or H ϩ (15,18), utilizing the free energy from the downhill translocation of one cosubstrate to drive uphill translocation of the other (3,39,40,42,44), and all three cations compete for a common binding pocket (6,18,26,31). A threading model of MelB (45) based on the crystal structure of LacY (2,16,17,30) suggests that MelB is a member of the major facilitator superfamily; thus, the protein is likely organized into two pseudosymmetrical six-helix bundles connected by a long middle loop surrounding an internal cavity facing the cytoplasm.…”

“…1) (11,15,43,45). Helix IV is in the center of a charge/H-bond network involved in the binding of the two cosubstrates (4,36,45,47).…”

“…Gly117 in MelB St has been mutated previously to Ala, Pro, Trp, or Arg (15), and the effects of these mutations on cosubstrate binding and transport depend on the physical and chemical properties of the side chain. Compared to wild-type (WT) MelB St , the G117A mutant exhibits little difference in either cosubstrate binding or Na ϩ -or Li ϩ -coupled melibiose transport; the other three mutations reduce melibiose active transport and decrease the apparent affinity for cations, with a stronger effect on Na ϩ .…”