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• W2012034385 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 multidrug-binding pocket. 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. Multidrug resistance is the phenomenon in which a single transmembrane transport protein or protein complex mediates the export of a wide range of structurally dissimilar toxic compounds. Multidrug efflux pumps are ubiquitous and belong to five distinct transport protein families, including the ATP-binding cassette superfamily, the major facilitator superfamily (MFS) 3The abbreviations used are: MFSmajor facilitator superfamilyDAPI4′,6-diamidino-2-phenylindoleDddiamidinodiphenylamideEtethidiumMICminimum inhibitory concentration(s)NEMN-ethyl-maleimideQacquaternary ammonium compound(s)TMStransmembrane segment(s). 3The abbreviations used are: MFSmajor facilitator superfamilyDAPI4′,6-diamidino-2-phenylindoleDddiamidinodiphenylamideEtethidiumMICminimum inhibitory concentration(s)NEMN-ethyl-maleimideQacquaternary ammonium compound(s)TMStransmembrane segment(s)., the resistance/nodulation/cell division superfamily, the drug/metabolite transporter family, and the multidrug and toxic compound extrusion family (1Saier Jr., M.H. Paulsen I.T. Semin. Cell Dev. Biol. 2001; 12: 205-213Crossref PubMed Scopus (280) Google Scholar). These proteins have increasingly become a major obstacle for the treatment of bacterial infectious diseases and the chemotherapy of human cancers. A detailed understanding of the substrate recognition and transport mechanisms of these transporters is required to overcome the problems associated with multidrug resistance. major facilitator superfamily 4′,6-diamidino-2-phenylindole diamidinodiphenylamide ethidium minimum inhibitory concentration(s) N-ethyl-maleimide quaternary ammonium compound(s) transmembrane segment(s). major facilitator superfamily 4′,6-diamidino-2-phenylindole diamidinodiphenylamide ethidium minimum inhibitory concentration(s) N-ethyl-maleimide quaternary ammonium compound(s) transmembrane segment(s). The multidrug resistance gene qacA is carried by multiresistance plasmids from clinical isolates of Staphylococcus aureus and other coagulase-negative staphylococci (2Lyon B.R. Skurray R. Microbiol. Rev. 1987; 51: 88-134Crossref PubMed Google Scholar, 3Leelaporn A. Paulsen I.T. Tennent J.M. Littlejohn T.G. Skurray R.A. J. Med. Microbiol. 1994; 40: 214-220Crossref PubMed Scopus (133) Google Scholar). qacA encodes a 514-amino-acid protein, QacA, that possesses 14 α-helical transmembrane segments (TMS) (4Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3630-3635Crossref PubMed Scopus (234) Google Scholar). QacA is a member of the MFS and mediates substrate/H+ antiport utilizing the proton motive force as the driving force (5Littlejohn T.G. Paulsen I.T. Gillespie M.T. Tennent J.M. Midgley M. Jones I.G. Purewal A.S. Skurray R.A. FEMS Microbiol. Lett. 1992; 95: 259-266Crossref Google Scholar). It confers resistance to >30 structurally dissimilar organic cations, including monovalent cationic compounds, such as quaternary ammonium compounds (Qacs) and intercalating dyes, and bivalent compounds, such as diamidines and biguanidines (6Mitchell B.A. Brown M.H. Skurray R.A. Antimicrob. Agents Chemother. 1998; 42: 475-477Crossref PubMed Scopus (110) Google Scholar, 7Brown M.H. Skurray R.A. J. Mol. Microbiol. Biotechnol. 2001; 3: 163-170PubMed Google Scholar). Fluorimetric analysis of QacA-mediated export suggests that monovalent and bivalent substrates bind to distinct sites on the QacA protein (8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Comparative and mutagenesis studies of QacA and the closely related QacB protein, which differs from QacA by conferring little or no resistance to bivalent compounds, viz. diamidines and biguanidines, have revealed that the presence of an acidic residue at position 323 in TMS 10 of QacA is essential for the high levels of resistance seen to bivalent compounds (4Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3630-3635Crossref PubMed Scopus (234) Google Scholar). Competition studies show that QacA utilizes a high affinity binding site(s) for the transport of bivalent substrates that are absent in QacB, implying direct involvement of Asp-323 and hence TMS 10 in the substrate recognition process (7Brown M.H. Skurray R.A. J. Mol. Microbiol. Biotechnol. 2001; 3: 163-170PubMed Google Scholar, 8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To date, very limited functional and structural information of the 14-TMS MFS proteins is available, and crystallographic studies of these proteins are inherently difficult to perform, necessitating molecular and biochemical studies of these proteins. Because QacA contains no intrinsic cysteine (Cys) residues (see Fig. 1), it is an ideal target for Cys-scanning mutagenesis studies (9Loo T.W. Clarke D.M. Biochim. Biophys. Acta. 1999; 1461: 315-325Crossref PubMed Scopus (88) Google Scholar, 10Tamura N. Konishi S. Iwaki S. Kimura-Someya T. Nada S. Yamaguchi A. J. Biol. Chem. 2001; 276: 20330-20339Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 11Frillingos S. Sahin-Tóth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (319) Google Scholar). Therefore, to understand the importance of TMS 10 in QacA-mediated efflux, Cys-scanning mutagenesis was carried out on TMS 10 and its flanking regions. The effect of Cys substitution on protein expression was studied by Western blot analysis. The extents of TMS 10 were examined by analyzing the relative reactivity of the QacA Cys-substituted mutant proteins to two differently labeled maleimido compounds, and functionally important residues were identified by phenotypic analysis and fluorimetric transport assays. Bacterial Strains—The Escherichia coli strain DH5α (12Hanahan D. J. Mol. Biol. 1983; 166: 557-580Crossref PubMed Scopus (8138) Google Scholar) was used as the host strain for general cloning purposes, Western blotting, and solvent accessibility analyses. The E. coli strain BHB2600 (13Hohn B. Methods Enzymol. 1979; 68: 299-309Crossref PubMed Scopus (350) Google Scholar) was used for minimum inhibitory concentration (MIC) analyses and fluorimetric transport assays. Both strains were cultured at 37 °C in Luria broth containing 100 μg/ml ampicillin where appropriate. Chemicals—Benzalkonium, chlorhexidine, 4′,6-diamidino-2-phenylindole (DAPI), dequalinium, ethidium (Et), pyronin Y, and N-ethylmaleimide (NEM) were purchased from Sigma; [14C]NEM from PerkinElmer Life Sciences; and fluorescein-5-maleimide from Molecular Probes. Diamidinodiphenylamine (Dd) was provided by Rhône-Poulenc Rorer (Dagenham, UK). All other materials were of reagent grade and obtained from commercial sources. Site-directed Mutagenesis—Site-directed mutants were constructed per the QuikChange™ technique (Stratagene) using pairs of mutagenic primers and plasmid pSK4322 as a template. This plasmid was constructed by cloning a PCR-derived 1.6-kb qacA-coding fragment from plasmid pSK1 (14Rouch D.A. Cram D.S. DiBerardino D. Littlejohn T.G. Skurray R.A. Mol. Microbiol. 1990; 4: 2051-2062Crossref PubMed Scopus (239) Google Scholar) into the EcoRI and BamHI sites of the vector pBluescript II SK(+). A His6 tag was also incorporated into the C terminus of QacA during PCR. All mutated sequences were verified by DNA sequencing. Labeling of the QacA Cys Mutant Proteins with Fluorescein Maleimide or [14C]NEM—E. coli DH5α cells harboring plasmids encoding QacA Cys-substituted mutants were inoculated into Luria broth supplemented with 100 μg/ml ampicillin, grown to OD650 = 0.9 and collected by centrifugation. Bacterial cells were disrupted by passing three times through a French pressure cell (SLM Aminco, Spectronic Instruments) at 20,000 psi. After removal of cell debris by centrifugation, membrane vesicles were collected by ultracentrifugation at 125,000 × g for 1 h and resuspended in 50 mm Tris-HCl buffer (pH 7.0) to a protein concentration of 20 mg/ml. Typically, 30 μl of membrane vesicles were used for each reaction. Fluorescein maleimide or [14C]NEM was added to a final concentration of 0.25 mm and then incubated at 30 °C for 10 min. The reactions were terminated by adding NEM to a final concentration of 20 mm. The membrane vesicles were solubilized with 1% SDS and QacA mutant proteins purified by affinity chromatography using Ni2+ nitrilotriacetic acid-agarose. Approximately 2 μg of the purified QacA protein was subjected to SDS-PAGE. For fluorescein maleimide labeling, fluorescein maleimide bound to QacA was visualized by scanning wet gels directly using a Molecular Imager® FX (Bio-Rad). The gels were then stained with Coomassie Brilliant Blue R-250 and scanned with a GS-710 calibrated imaging densitometer (Bio-Rad). For [14C]NEM labeling, the gel was first stained and dried. The dried gel was exposed to a phosphor screen, and the [14C]NEM-bound QacA mutant proteins were visualized by scanning with a Molecular Imager® FX (Bio-Rad). To determine the effect of Et and DAPI binding on the labeling of the QacA Cys mutant proteins by fluorescein maleimide, membrane vesicles containing QacA mutant proteins were first incubated at 30 °C for 5 min in the absence or presence of 1 mm Et or DAPI; labeling of mutant proteins was performed as described above. Determination of Resistance Profiles and Fluorimetric Analysis of QacA-mediated Et and DAPI Efflux—MIC analyses and transport assays of QacA-mediated efflux of Et and DAPI were performed as described previously (8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Briefly, transport activities of bacterial cells incubated with 12 different concentrations of each substrate were measured fluorimetrically. Initial transport velocities were calculated by averaging the linear part of each fluorescence-decreasing curve and computationally fitted to estimated Vmax and Km values. Vmax values of each QacA mutant protein were normalized against the protein expression levels obtained by densitometric scanning of Western blots using a QacA-specific antiserum. Effect of NEM Treatment on the Efflux of Et and DAPI—Aliquots of bacterial cells prepared as previously described (8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) were incubated in the presence or absence of 5 mm NEM at 37 °C for 20 min. After removal of NEM by washing with 20 mm HEPES buffer (pH 7.0), the cells were resuspended in the above buffer, loaded with 15 μm Et or 10 μm DAPI, and the QacA-mediated efflux analyzed as previously described (8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Second-site Suppressor Analysis of the QacA D323C Mutant—To isolate second-site suppressors, E. coli DH5α cells harboring the plasmid encoding the QacA D323C mutant were plated on Luria broth agar plates supplemented with a non-permissive concentration of the bivalent compound Dd (100 μg/ml) and incubated at 37 °C for 48 h. The resulting colonies were purified, their plasmids extracted and transformed into fresh E. coli DH5α cells, and the resistance profiles of the resulting transformants determined by MIC analysis. The 1.6-kb qacA-coding regions of the plasmids able to confer resistance to Dd were then fully sequenced. Cys-scanning Mutagenesis of QacA TMS 10 and the Expression of Mutant Proteins—Previous studies revealed that a negatively charged Asp at position 323 of TMS 10 of QacA was essential for the recognition of bivalent cationic compounds, implying the direct involvement of this TMS in the substrate recognition process (4Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3630-3635Crossref PubMed Scopus (234) Google Scholar, 8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). To further understand the importance of this TMS in the function of QacA, 35 amino acid residues in putative TMS 10 and its flanking regions, from Ser-308 to Val-342 of QacA (Fig. 1), were replaced with Cys individually by site-directed mutagenesis and the resulting mutants functionally and biochemically characterized. Western blotting with a QacA-specific anti-serum revealed that most of the QacA mutant proteins were expressed at levels essentially comparable with that of the QacA wild-type protein (data not shown), indicating that Cys substitution generally did not have a significant effect on the expression of the mutant proteins. However, the QacA mutant proteins K311C, R336C, and K340C were expressed at levels below or equivalent to 50% of that of QacA wild-type protein, suggesting that these basic residues may be important for protein expression, insertion, and/or stability. Examination of the Extents of QacA TMS 10—Although the rough boundaries of TMS 10 had been predicted by hydropathy analysis and confirmed experimentally using limited gene fusions to both alkaline phosphatase and β-galactosidase (4Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3630-3635Crossref PubMed Scopus (234) Google Scholar), the exact limits were not clear. Such information can be obtained by determining the reactivity of Cys-substituted mutant proteins with maleimide derivatives (10Tamura N. Konishi S. Iwaki S. Kimura-Someya T. Nada S. Yamaguchi A. J. Biol. Chem. 2001; 276: 20330-20339Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 15Kimura T. Suzuki M. Sawai T. Yamaguchi A. Biochemistry. 1996; 35: 15896-15899Crossref PubMed Scopus (39) Google Scholar, 16Poelarends G.J. Konings W.N. J. Biol. Chem. 2002; 277: 42891-42898Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Maleimido compounds react with a thiolate more readily than with a thiol group; therefore, they selectively react with Cys residues that are exposed to an aqueous medium (15Kimura T. Suzuki M. Sawai T. Yamaguchi A. Biochemistry. 1996; 35: 15896-15899Crossref PubMed Scopus (39) Google Scholar), whereas Cys residues in a hydrophobic environment are expected to have a much lower reactivity. In this study, the relative reactivities of the QacA Cys-substituted mutant proteins to maleimide derivatives were determined using both fluorescein maleimide and [14C]NEM. The results using these two differently labeled maleimido compounds were in good agreement; hence only experiments using fluorescein maleimide were performed in detail, the results of which are shown in Fig. 2. The QacA mutant proteins S308C and P309C were strongly reactive with fluorescein maleimide, whereas mutant proteins F310C and K311C were only weakly reactive. Cys residues at positions 312–329 were barely reactive with fluorescein maleimide, except for A327C, suggesting that the stretch of 20 amino acid residues from Phe-310 to Ile-329 forms the hydrophobic core of the transmembrane helix. The majority of the QacA mutant proteins from A330C to I341C were reactive with fluorescein maleimide but to significantly different extents, with only the A335C and G338C mutant proteins being strongly reactive (Fig. 2). It is likely that residues Ala-330–Ile-341 form some kind of structure or are partially embedded in the membrane bilayer and are therefore not completely exposed to the aqueous medium and hence cannot efficiently react with fluorescein maleimide. The QacA V342C mutant protein did not react with fluorescein maleimide, suggesting that position 342 is located in a hydrophobic environment and probably lies within TMS 11, a result in agreement with the originally predicted model (Fig. 1). Minimum Inhibitory Concentration Analysis of the QacA Mutants—To determine the functional importance of the studied residues, resistance profiles of the QacA mutants were determined by MIC analysis using substrates from different chemical classes, including the monovalent compounds Et, pyronin Y, and benzalkonium and the bivalent compounds dequalinium, DAPI, Dd, and chlorhexidine. The results showed that the majority of the mutants conferred resistance to most of the substrates tested at levels comparable with those of QacA wild type (Fig. 3). However, some QacA derivatives, such as L314C, Y315C, G322C, and A327C, were more susceptible to one or more substrates tested but not to an entire category of monovalent or bivalent substrates. The QacA G313C mutant conferred significantly reduced resistance to dequalinium and chlorhexidine and no resistance to all of the other compounds tested, and the QacA D323C mutant lost resistance to all of the bivalent compounds tested, indicating that Gly-313 is important for resistance to all QacA substrates, whereas Asp-323 is essential only for resistance to bivalent compounds, as previously reported (4Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3630-3635Crossref PubMed Scopus (234) Google Scholar, 8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Et and DAPI Efflux by QacA Mutants—To verify the phenotypes conferred by these QacA mutants, fluorimetric analyses of the efflux of Et and DAPI, which were chosen as representatives of monovalent and bivalent substrates of QacA, respectively, were performed as previously described (8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), and the kinetic parameters of transport, Vmax (Fig. 4) and Km (data not shown), determined. The results showed that only one of the 35 mutants, the QacA G313C mutant, completely lost Et export activity (Fig. 4A). The remaining QacA mutants mediated significant Et efflux, with activities ranging from ∼48 (M324C) to 231% (R336C) compared with that of the QacA wild type. For DAPI export, both the QacA G313C and D323C mutants completely lost DAPI transport activity. These results further confirmed the importance of residues Gly-313 and Asp-323 in efflux mediated by QacA. Additionally, replacement of Met-319 with Cys resulted in a significant reduction in DAPI transport activity (∼12% of that of QacA wild type; Fig. 4B), indicating that this residue was also important for DAPI transport. The other QacA mutants retained significant DAPI transport activities (Fig. 4B). Transport analyses of the QacA K311C, R336C, and K340C mutants revealed that these derivatives have high Et and DAPI transport activities when taking into account their low expression levels (Fig. 4). It is possible that the reduced amount of these mutant proteins is an adjustment made by the cell so that these highly active derivatives are not present at toxic levels. The Km values of Et and DAPI transport by the QacA mutants were all <20 μm (data not shown), consistent with previous results (8Mitchell B.A. Paulsen I.T. Brown M.H. Skurray R.A. J. Biol. Chem. 1999; 274: 3541-3548Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), indicating that QacA interacts with Et and DAPI specifically and these amino acid substitutions do not greatly affect substrate binding. Effect of NEM on Et and DAPI Efflux by QacA Mutants—NEM has widely been used to probe the substrate-binding site/transport pathway of membrane transport proteins (11Frillingos S. Sahin-Tóth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (319) Google Scholar, 17Kimura-Someya T. Iwaki S. Yamaguchi A. J. Biol. Chem. 1998; 273: 32806-32811Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). For example, residues in the E. coli lactose permease LacY, which have Cys-substituted mutants inactivated by NEM alkylation, are often found to be in close vicinity to the substrate-binding site (18Frillingos S. Kaback H.R. Protein Sci. 1997; 6: 438-443Crossref PubMed Scopus (45) Google Scholar, 19Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1217) Google Scholar, 20Kaback H.R. Sahin-Tóth M. Weinglass A.B. Nat. Rev. Mol. Cell Biol. 2001; 2: 610-620Crossref PubMed Scopus (249) Google Scholar). To test whether any of the residues in TMS 10 of QacA were likely to form or be spatially juxtaposed to the substrate-binding site(s)/transport pathway, the effects of NEM treatment on Et and DAPI efflux mediated by the QacA mutants were examined. The QacA G313C and D323C derivatives lost both Et and DAPI or DAPI transport activities, respectively, and hence could not be tested. Results showed that there were no mutants with an Et efflux capability that was completely abolished by the addition of NEM (Fig. 5A). However, Et efflux mediated by the QacA P309C, A320C, and A327C derivatives was inhibited significantly (≥50%) by ∼65, 50, and 70%, respectively, whereas Et efflux by the QacA A334C mutant was enhanced to 250% (Fig. 5A). The magnitudes of NEM inhibition of the above mutants were similar over a range of NEM concentrations (1–10 mm) or different incubation time periods (10–40 min) in the presence of 5 mm NEM (data not shown), indicating that the incomplete inhibition observed was not due to an incomplete reaction of the Cys residues with NEM. Therefore, the inhibition of QacA-mediated Et efflux by NEM was probably because of the steric hindrance of substrate binding or translocation resulting from NEM alkylation, as suggested using similar studies that have been performed with other transport proteins (17Kimura-Someya T. Iwaki S. Yamaguchi A. J. Biol. Chem. 1998; 273: 32806-32811Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 18Frillingos S. Kaback H.R. Protein Sci. 1997; 6: 438-443Crossref PubMed Scopus (45) Google Scholar, 19Abramson J. Smirnova I. Kasho V. Verner G. Kaback H.R. Iwata S. Science. 2003; 301: 610-615Crossref PubMed Scopus (1217) Google Scholar). The effect of NEM treatment on the DAPI efflux of the QacA mutants was also examined (Fig. 5B). NEM treatment abolished DAPI efflux mediated by the QacA P309C mutant and significantly enhanced that by the QacA A334C mutant by ∼10-fold (Fig. 5B). No significant effect of NEM treatment on the DAPI efflux by the other QacA mutants was observed. Binding of Et and DAPI Induced a Different Conformational Change to QacA—To examine whether any amino acids within TMS 10 of QacA were so close to the substrate-binding site(s) that the introduced Cys residues could be allosterically protected from maleimide alkylation by the addition of substrates, or whether substrate binding induced a conformational change of the QacA protein, thereby making the Cys residues more or less accessible to solvent, the effects of pre-incubation with Et and DAPI on the labeling of a number of QacA mutant proteins by fluorescein maleimide, including P309C, L316C, A320C, D323C, M324C, A327C, P328C, A330C, P331C, G332C, A334C, and F337C, were analyzed. Most of the mutants tested possessed a Cys residue predicted to be on the same face of TMS 10 as Asp-323, which was likely to be close to the substrate-binding site, or with relatively high solvent accessibility (Fig. 2), could be tested for substrate protection against maleimide alkylation. The results indicated that, although pre-incubation with Et had no significant effect on the maleimide labeling of all of the mutant proteins tested (data not shown), pre-incubation with DAPI did significantly increase the labeling of mutant proteins A327C and P331C by ∼4.2- and 6.7-fold, respectively (Fig. 6). This suggested that the binding of DAPI may have caused a conformational change of the QacA protein, rendering residues P331C and A327C more exposed to the aqueous phase and hence allowing higher degrees of alkylation by fluorescein maleimide. Isolation and Characterization of a QacA D323C/A320E Double Mutant—To determine whether an additional mutation can compensate for the loss of the negative charge at position 323 of TMS 10, a second-site suppressor mutant of the QacA D323C mutant was isolated. After growth on a non-permissive concentration of the substrate Dd, only one resistant QacA mutant was isolated. DNA sequencing of the promoter and coding region of qacA confirmed that the initial Cys substitution was still present in addition to a second-site mutation, creating a QacA D323C/A320E double mutant. This double mutant regained high levels of resistance to bivalent compounds (Fig. 3) and displayed a much higher DAPI transport activity, ∼10-fold higher than that of the QacA wild type, whereas it retained similar Et transport activity as the QacA wild type (data not shown). This indicated that an acidic Glu residue at position 320 of TMS 10, which is one turn away from position 323 and on the same face of the helix (Fig. 7), can compensate for the mutation of D323C. When treated with NEM, DAPI efflux mediated by this mutant was completely abolished, suggesting that Asp-323 is directly involved in bivalent substrate binding. Phenotypic and fluorimetric analyses of the QacA Cys mutants showed that the majority of the residues were not crucial for the function of QacA (Figs. 3 and 4). However, some Cys substitutions exerted substrate-specific effects; for example, the QacA L314C and Y315C mutants lost resistance to benzalkonium but conferred substantial resistance to other compounds (Fig. 3). Such altered substrate specificity, because of the mutation of a single amino acid, has been well documented for multidrug resistance proteins (4Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3630-3635Crossref PubMed Scopus (234) Google Scholar, 21Loo T.W. Clarke D.M. Biochemistry. 1994; 33: 14049-14057Crossref PubMed Scopus (125) Google Scholar, 22Klyachko K.A. Schuldiner S. Neyfakh A.A. J. Bacteriol. 1997; 179: 2189-2193Crossref PubMed Scopus (67) Google Scholar, 23Taguchi Y. Kino K. Morishima M. Komano T. Kane S.E. Ueda K. Biochemistry. 1997; 36: 8883-8889Crossref PubMed Scopus (56) Google Scholar, 24Edgar R. Bibi E. EMBO J. 1999; 18: 822-832Crossref PubMed Scopus (125) Google Scholar) and reflects the poly-specific nature of the multidrug recognition mechanism. By contrast, the QacA D323C mutant lost resistance to all tested bivalent compounds and DAPI efflux capability but retained wild-type levels of resistance to monovalent compounds and mediated Et efflux to a level not significantly different from that of the QacA wild type (Figs. 3 and 4), substantiating the importance of this residue in the recognition of bivalent compounds (4Paulsen I.T. Brown M.H. Littlejohn T.G. Mitchell B.A. Skurray R.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3630-3635Crossref PubMed Scopus (234) Google Scholar, 8Mitchell B.A. Paulsen I.T. Brown M" @default.
• W2012034385 created "2016-06-24" @default.
• W2012034385 creator A5026897209 @default.
• W2012034385 creator A5028978450 @default.
• W2012034385 creator A5033626038 @default.
• W2012034385 creator A5079254981 @default.
• W2012034385 date "2006-01-01" @default.
• W2012034385 modified "2023-09-29" @default.
• W2012034385 title "Role of Transmembrane Segment 10 in Efflux Mediated by the Staphylococcal Multidrug Transport Protein QacA" @default.
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