Role of transmembrane segment 10 in efflux mediated by the staphylococcal multidrug transport protein QacA.
The staphylococcal multidrug exporter QacA confers resistance to a wide range of structurally dissimilar monovalent and bivalent cationic antimicrobial compounds. To understand the functional importance of transmembrane segment 10, which is thought to be involved in substrate binding, cysteine-scanning mutagenesis was performed in which 35 amino acid residues in the putative transmembrane helix and its flanking regions were replaced in turn with cysteine. Solvent accessibility analysis of the introduced cysteine residues using fluorescein maleimide indicated that transmembrane segment 10 of QacA contains a 20-amino-acid hydrophobic core and may extend from Pro-309 to Ala-334. Phenotypic analysis and fluorimetric transport assays of these mutants showed that Gly-313 is important for the efflux of both monovalent and bivalent cationic substrates, whereas Asp-323 is only important for the efflux of bivalent substrates and probably forms part of the bivalent substrate-binding site(s) together with Met-319. Furthermore, the effects of N-ethyl-maleimide treatment on ethidium and 4',6-diamidino-2-phenylindole export mediated by the QacA mutants suggest that the face of transmembrane segment 10 that contains Asp-323 may also be close to the monovalent substrate-binding site(s), making this helix an integral component of the QacA multidrug-binding pocket.
Xu
Zhiqiang
Z
School of Biological Sciences, University of Sydney, New South Wales, Australia.
O'Rourke
Brendon A
BA
Skurray
Ronald A
RA
Brown
Melissa H
MH
D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry*; D001665:Binding Sites; D001692:Biological Transport; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D018432:Drug Resistance, Multiple; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008301:Maleimides / Q000737:chemistry; D026901:Membrane Transport Proteins / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D010641:Phenotype; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012997:Solvents / Q000737:chemistry; D013211:Staphylococcus aureus / Q000378:metabolism*
D001426:Bacterial Proteins; D008301:Maleimides; D026901:Membrane Transport Proteins; D012997:Solvents; C068498:qacA protein, Staphylococcus aureus; C043592:maleimide; D001224:Aspartic Acid; D008715:Methionine; D003545:Cysteine