Role of Transmembrane Segment 10 in Efflux Mediated by the Staphylococcal Multidrug Transport Protein QacA

Abstract

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 multidrugbinding pocket.

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

“…In two other well characterized but more distant MFS multidrug antiporters from Gram-positive bacteria, LmrP and QacA, membrane-embedded carboxylic acids in various TMs have been shown to play a role in substrate binding and proton translocation, although the location of the carboxylic amino acids is not conserved (62)(63)(64)(65)(66). Among the well characterized MFS antiporters, the highest similarity and, presumably, mechanism is indeed observed between BbMAT and rVMAT2 (Fig.…”

Analysis of Tryptophan Residues in the Staphylococcal Multidrug Transporter QacA Reveals Long-Distance Functional Associations of Residues on Opposite Sides of the Membrane

“…Transport of the monovalent substrate ethidium and transport of the bivalent substrate DAPI were measured fluorimetrically in whole E. coli cells as previously described (22,32). Briefly, 1-ml volumes of cells expressing QacA or a QacA derivative, suspended in 20 mM HEPES (pH 7.0) with an OD 600 of 0.6, were loaded with either 15 M ethidium or 8 M DAPI in the presence of 10 mM carbonyl cyanide m-chlorophenylhydrazone.…”

“…E. coli strain DH5␣ [supE44 ⌬lacU169 (80lacZ⌬M15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1] (8) was used as the host for all cloning procedures and functional analyses. The plasmids used were pBluescript II SK (Stratagene); the pBluescript II SK-based qacA clone pSK4322 (32), which encodes QacA protein with a C-terminal hexahistidine tag; and a pSK4322 derivative, pSK7400, that encodes a QacA W58A mutant (this study). Bacterial strains were cultured in Luria-Bertani broth and on LuriaBertani agar plates, unless otherwise stated.…”

“…Western blotting was conducted on lysates of E. coli cells expressing wild-type QacA or variants by using an anti-QacA rabbit polyclonal antiserum as described previously (32). Western blot membranes were scanned using a GS-710 calibrated imaging densitometer (Bio-Rad), and results were analyzed using Quantity One software (Bio-Rad) to determine the level of expression of each QacA variant relative to that of the wild-type protein.…”

“…1), which displays exceptionally broad substrate specificity, conferring resistance to both monovalent and bivalent cationic antimicrobials from at least 12 different chemical families (5). Competition studies of the QacA transporter have determined that monovalent and bivalent cationic substrates interact with QacA at distinct sites (22); however, recent results have suggested that these sites are in physical proximity, possibly within the same extended binding region (32). Additionally, as observed for other multidrug transporters, the spatial organization of amino acid side chains that mediate stabilizing interactions with drugs within the QacA binding region appears to be flexible (3,11).…”