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

Department of Cell Physiology & Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA.
Biochemistry (Impact Factor: 3.02). 03/2012; 51(13):2950-7. DOI: 10.1021/bi300230h
Source: PubMed


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.

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    ABSTRACT: The melibiose permease of Salmonella enterica serovar Typhimurium (MelB(St)) catalyzes symport of melibiose with Na(+), Li(+), or H(+). Bioinformatics and mutational analyses indicate that a conserved Gly117 (helix IV) is a component of the Na(+)-binding site. In this study, Gly117 was mutated to Ser, Asn, or Cys. All three mutations increase the maximum rate (V(max)) for melibiose transport in Escherichia coli DW2 and greatly decrease Na(+) affinity, indicating that intracellular release of Na(+) is facilitated. Rapid melibiose transport, particularly by the G117N mutant, triggers osmotic lysis in the lag phase of growth. The findings support the previous conclusion that Gly117 plays an important role in cation binding and translocation. Furthermore, a spontaneous second-site mutation (P148L between loop(4-5) and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases V(max) with little effect on cosubstrate binding in G117C, G117S, and G117N mutants. Thus, the P148L mutation specifically inhibits transport velocity and thereby blocks the lethal effect of elevated melibiose transport in the Gly117 mutants.
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