Objectives: To elucidate the importance of a region in QacA predicted to be important in antimicrobial substrate recognition.
Methods: A total of 38 amino acid residues within or flanking putative transmembrane helix segment (TMS) 12 of QacA were individually replaced with cysteine using site-directed mutagenesis. The impact of these mutations on protein expression, drug resistance, transport activity and interaction with sulphhydryl-binding compounds was determined.
Results: Accessibility analysis of cysteine-substituted mutants identified the extents of TMS 12, which allowed for refinement of the QacA topology model. Mutation of Gly-361, Gly-379 and Ser-387 in QacA resulted in reduced resistance to at least one bivalent substrate. Interaction with sulphhydryl-binding compounds in efflux and binding assays demonstrated the role of Gly-361 and Ser-387 in the binding and transport pathway of specific substrates. The highly conserved residue Gly-379 was found to be important for the transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and interhelical interactions.
Conclusions: TMS 12 and its external flanking loop is required for the structural and functional integrity of QacA and contains amino acids directly involved in the interaction with substrates.
- bacterial protein
- biological transport
- QacA protein