Role of a conserved membrane-embedded acidic residue in the multidrug transporter MdfA

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• The multidrug transporter MdfA from Escherichia coli plays a crucial role in effluxing various compounds.
• A negatively charged residue, Glutamate 26 (Glu26), is proposed to be vital for substrate recognition.
• Understanding residue roles is key to characterizing transporter mechanisms and specificity.

Purpose of the Study:

• To investigate the role of the Glu26 residue in the substrate recognition and transport mechanism of MdfA.
• To determine the impact of varying chemical properties at position 26 on MdfA's substrate specificity.

Main Methods:

• Site-directed mutagenesis was used to create various replacements for the Glu26 residue in MdfA.
• Transport assays were performed using neutral and positively charged substrates to characterize transporter activity.
• Analysis focused on the tolerance of MdfA to changes in size, hydrophobicity, and charge at position 26.

Main Results:

• MdfA demonstrated significant tolerance to diverse substitutions at position 26, including changes in size, hydrophobicity, and charge, for neutral substrates.
• While a negative charge at position 26 facilitates the transport of positively charged substrates, its absence did not always abolish interaction with cationic drugs.
• These findings challenge the notion of highly defined binding pockets in multidrug transporters.

Conclusions:

• The substrate recognition pocket of MdfA exhibits remarkable structural and chemical promiscuity.
• This flexibility in the binding site likely contributes to the broad substrate specificity observed in multidrug transporters.
• The role of specific charged residues in substrate binding may be less rigid than previously assumed.