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

Department of Cell Physiology & Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
Journal of bacteriology (Impact Factor: 2.81). 08/2012; 194(20):5538-44. DOI: 10.1128/JB.01206-12
Source: PubMed


The melibiose permease of Salmonella enterica serovar Typhimurium (MelBSt) 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 (Vmax) 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 loop4-5 and helix V) in the G117C mutant prevents cell lysis. This mutation significantly decreases Vmax 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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    ABSTRACT: The bacterial melibiose permease (MelB) belongs to the glycoside-pentoside-hexuronide:cation symporter family, a part of the major facilitator superfamily (MFS). Structural information regarding glycoside-pentoside-hexuronide:cation symporter family transporters and other Na(+)-coupled permeases within MFS has been lacking, although a wealth of biochemical and biophysical data are available. Here we present the three-dimensional crystal structures of Salmonella typhimurium MelBSt in two conformations, representing an outward partially occluded and an outward inactive state of MelBSt. MelB adopts a typical MFS fold and contains a previously unidentified cation-binding motif. Three conserved acidic residues form a pyramidal-shaped cation-binding site for Na(+), Li(+) or H(+), which is in close proximity to the sugar-binding site. Both cosubstrate-binding sites are mainly contributed by the residues from the amino-terminal domain. These two structures and the functional data presented here provide mechanistic insights into Na(+)/melibiose symport. We also postulate a structural foundation for the conformational cycling necessary for transport catalysed by MFS permeases in general.
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    ABSTRACT: The crystal structure of the Na+-coupled melibiose permease of Salmonella enterica serovar Typhimurium (MelBSt) demonstrates that MelB is a member of the major facilitator superfamily of transporters. Arg residues at positions 295, 141, and 363 are involved in interdomain interactions at the cytoplasmic side by governing three clusters of electrostatic/polar interactions. Insertion of (one at a time) Glu, Leu, Gln, or Cys at positions R295, R141, and R363, or Lys at position R295, inhibits active transport of melibiose to a level of 2 to 20% of the value for wild-type (WT) MelBSt, with little effect on binding affinities for both sugar and Na+. Interestingly, a spontaneous suppressor, D35E (periplasmic end of helix I), was isolated from the R363Q MelBSt mutant. Introduction of the D35E mutation in each of the mutants at R295, R141 (except R141E), or R363 rescues melibiose transport to up to 91% of the WT value. Single-site mutations for the pair of D35 and R175 (periplasmic end of helix VI) were constructed by replacing Asp with Glu, Gln, or Cys and R175 with Gln, Asn, or Cys. All mutants with mutations at R175 are active, indicating that a positive charge at R175 is not necessary. Mutant D35E shows reduced transport; D35Q and D35C are nearly inactivated. Surprisingly, the D35Q mutation partially rescues both R141C and R295Q mutations. The data support the idea that Arg at position 295 and a positive charge at positions 141 and 363 are required for melibiose transport catalyzed by MelBSt, and their mutation inhibits conformational cycling, which is suppressed by a minor modification at the opposite side of the membrane.
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