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Updated: Aug 3, 2026
Chronic Salmonella Infected Mouse Model
Published on: May 31, 2010
Suppression of conformation-compromised mutants of Salmonella enterica serovar Typhimurium MelB
Anowarul Amin1, Abdul S Ethayathulla1, Lan Guan2
1Department of Cell Physiology and Molecular Biophysics, Center for Membrane Protein Research, Texas Tech University Health Sciences Center, Lubbock, Texas, USA.
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.
Insights
Key arginine residues in the melibiose permease (MelBSt) are crucial for active transport. Suppressor mutations can restore melibiose transport function, revealing insights into transporter conformational cycling.
Area of Science:
- Structural Biology
- Membrane Transport
- Biochemistry
Background:
- The melibiose permease (MelBSt) from Salmonella enterica serovar Typhimurium is a Na(+)-coupled transporter belonging to the major facilitator superfamily.
- Understanding the structural and functional roles of specific amino acid residues is critical for elucidating transporter mechanisms.
Purpose of the Study:
- To investigate the role of specific arginine residues (R295, R141, R363) in the Na(+)-coupled melibiose permease (MelBSt) function.
- To identify and characterize suppressor mutations that restore melibiose transport activity.
- To elucidate the mechanism of melibiose transport and conformational cycling in MelBSt.
Main Methods:
- Site-directed mutagenesis was employed to introduce specific amino acid substitutions in MelBSt.
- Melibiose transport assays were performed to quantify the activity of wild-type and mutant permeases.
- Binding affinities for melibiose and Na(+) were assessed.
- Suppressor mutations were isolated and characterized.
Main Results:
- Mutations at R295, R141, and R363 significantly inhibited melibiose transport while minimally affecting binding affinities.
- A spontaneous suppressor mutation, D35E, restored transport activity in mutants at R295, R141, and R363.
- Mutations at R175 did not abolish transport, indicating a positive charge is not essential at this position.
- The D35Q mutation also exhibited suppressive effects on certain MelBSt mutants.
Conclusions:
- Arginine residues at positions 295, 141, and 363 are essential for melibiose transport by MelBSt, likely by facilitating conformational cycling.
- Mutations in these residues inhibit transport by disrupting interdomain interactions.
- Suppressor mutations, particularly D35E, can restore transport function by compensating for the defects caused by mutations at the primary sites, suggesting communication across the transporter.
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