Suppression of conformation-compromised mutants of Salmonella enterica serovar Typhimurium MelB

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.

FIG 1

Overall fold of MelB_St. The N-terminal and C-terminal helix bundles are colored in green and blue, respectively. The structural information on MelB_St in this paper is based only on Mol-A of the PDB entry 4M64. The cytoplasmic loop_6-7, which contains two short helices (CH1 and CH2) and links the N- and C-terminal domains, is colored in yellow. The transmembrane helices are labeled with Roman numerals. (a) Side view. The positions involved in the interaction with Arg residues (295, 141, or 363) are shown by spheres (red, blue, and cyan for negative, positive, and other side chains, respectively). (b) Cytoplasmic view. The positions involved in the interaction with Arg residues (295, 141, or 363) are shown in sticks. All structure models are depicted using PyMOL.

FIG 2

Mutational analyses of the cytoplasmic Arg residues. (a) Twenty-five micrograms of crude membranes was loaded onto each lane of an SDS-16% PAGE gel. After transfer onto a polyvinylidene difluoride membrane, MelB_St proteins were detected with an anti-His tag antibody. (b and c) [³H]melibiose transport assays were carried out in E. coli DW2 intact cells expressing a given MelB_St mutant in 100 mM KP_i (pH 7.5) and 10 mM MgSO₄ at 0.7 mg/ml of protein. The transport assay was initiated by adding [³H]melibiose (0.4 mM, 10 mCi/mmol) in the absence or presence of 20 mM NaCl or LiCl. The initial rate (b), obtained by a linear fitting of melibiose uptake at 0, 5, and 10 s, and the level of melibiose accumulation (c) at 10 min, which represents the steady-state transport level, are expressed as percentages of the wild-type MelB_St values. Error bars show standard deviations (n = 2 to 3 for all mutants and n = 6 for the WT and DW2).

FIG 3

Revertants. (a) Melibiose fermentation. E. coli DW2 (ΔlacY ΔlacZ melA⁺ ΔmelB) cells were transformed with a plasmid encoding the WT, mutant R363Q, or double mutant R363Q/D35E; plated on MacConkey agar (lactose free) containing melibiose at 30 mM; and incubated at 37°C for 18 h. (b) Melibiose transport assay of the double mutants in the presence of 20 mM NaCl. (c) Transport assay of the triple mutants. Time course of [³H]melibiose transport with intact cells expressing WT MelB_St or a given mutant carried out as described in the legend to Fig. 2 and Materials and Methods. [³H]melibiose_in, intracellular melibiose. The single-site mutants are shown by green open diamonds, and corresponding second-site revertants are shown by filled blue triangles. Error bars show standard deviations (n = 2 for all mutants and n = 6 for the WT and DW2). (d) Membrane expression as described in the legend to Fig. 2.

FIG 4

Single D35 and R175 mutants. (a and b) Time courses of melibiose transport of D35 and R175 mutants. DW2 cells expressing either WT or a given MelB_St mutant were tested for Na⁺- and Li⁺-coupled melibiose transport as described in the legend to Fig. 2. [³H]melibiose_in, intracellular melibiose. Error bars show standard deviations (n = 2). (c) Western blot. Twenty-five micrograms of crude membranes was loaded onto each lane of an SDS-16% PAGE gel. MelB_St proteins were detected with an anti-His tag antibody. (d) Melibiose fermentation. The picture was taken after a 24-h incubation. Effect of D35 mutations on R141C and R295K mutants. (e) The melibiose transport assay was carried out as described in the legend to Fig. 2. Error bars show standard deviations (n = 2). Note that the y axis is scaled differently.