pH-induced Conformational Changes of AcrA, the Membrane Fusion Protein of Escherichia coli Multidrug Efflux System
2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês
10.1074/jbc.m305152200
ISSN1083-351X
AutoresHermia Ip, Kelly Stratton, Helen I. Zgurskaya, Jun Liu,
Tópico(s)Spectroscopy and Quantum Chemical Studies
ResumoThe multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates. The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates. Emergence of multidrug-resistant bacterial strains not only has hampered the current treatment of bacterial infections but also hindered the development of new therapeutic agents. Resistance mediated by multidrug efflux pumps as a major mechanism has been increasingly recognized. Available clinical data showed that 40–90% of some bacterial pathogens (Streptococcus pneumoniae, Streptococcus pyogenes, and Pseudomonas aeruginosa) bear efflux mechanisms for the major classes of available antibiotics (1.Brenwald N.P. Gill M.J. Wise R. Antimicrob. Agents Chemother. 1998; 42: 2032-2035Crossref PubMed Google Scholar, 2.Limia A. Jimenez M.L. Delgado T. Sanchez I. Lopez S. Lopez B. Rev. Esp. Quimioter. 1998; 11: 216-220PubMed Google Scholar, 3.Nikaido H. Clin. Infect. Dis. 1998; 27 (Suppl. 1, –S41): S32Crossref PubMed Scopus (288) Google Scholar, 4.Orden B. Perez T. Montes M. Martinez R. Pediatr. Infect. Dis. J. 1998; 17: 470-473Crossref PubMed Scopus (44) Google Scholar, 5.Shortridge V.D. Doern G.V. Brueggemann A.B. Beyer J.M. Flamm R.K. Clin. Infect. Dis. 1999; 29: 1186-1188Crossref PubMed Scopus (157) Google Scholar). Many drug efflux pumps have broad substrate specificity and expel a wide range of completely unrelated chemotherapeutic drugs. The AcrA-AcrB-TolC efflux system of Escherichia coli is such an example and is largely responsible for the intrinsic resistance of E. coli to most lipophilic antibiotics, detergents, and dyes (6.Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar, 7.Nikaido H. Zgurskaya H.I. J. Mol. Microbiol. Biotechnol. 2001; 3: 215-218PubMed Google Scholar). This system consists of a resistance-nodulation-cell division (RND) type efflux pump, AcrB, a periplasmic, membrane fusion protein (MFP), 1The abbreviations used are: MFPmembrane fusion proteinDTTdithiothreitolMES4-morpholineethanesulfonic acidHAhemagglutininMTSLmethanethiosulfonate spin labelSDSLsite-directed spin labelingDSCdifferential scanning calorimetry. AcrA, and a multifunctional outer membrane channel, TolC. Such organization allows the bacterium expel a wide variety of noxious compounds from the cell directly into the medium, bypassing the periplasm (8.Zgurskaya H.I. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7190-7195Crossref PubMed Scopus (304) Google Scholar). Similar multicomponent efflux systems have been found in other Gram-negative bacteria including P. aeruginosa, Enterobacter aerogenes, and Neisseria gonorrhoeae (3.Nikaido H. Clin. Infect. Dis. 1998; 27 (Suppl. 1, –S41): S32Crossref PubMed Scopus (288) Google Scholar, 6.Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar). membrane fusion protein dithiothreitol 4-morpholineethanesulfonic acid hemagglutinin methanethiosulfonate spin label site-directed spin labeling differential scanning calorimetry. Major progresses have been made for understanding the efflux mechanism in Gram-negative bacteria, which are highlighted by recent publications of the crystal structures of TolC and AcrB (9.Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar, 10.Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar, 11.Yu E.W. McDermott G. Zgurskaya H.I. Nikaido H. Koshland Jr., D. Science. 2003; 300: 976-980Crossref PubMed Scopus (338) Google Scholar). The TolC trimer comprises of two barrel-like structures joined together, the outer membrane β-barrel and the periplasmic α-barrel. The long α-barrel (∼100 Å) is thought to traverse the periplasm and interact with AcrB or inner membrane (9.Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar). Consistently, the AcrB protein, also as a trimer, contains two structural domains, the transmembrane domain (50 Å in thickness) and a headpiece that protrudes about 70 Å in depth into the periplasm (10.Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar). It is thought that AcrB and TolC may be directly docked with each other, forming a continuous pathway across the periplasm and the outer membrane. Substrates may gain access to the AcrB central cavity either from the cell interior through the transmembrane region, or from the periplasm through the vestibules of AcrB protein, which are then actively transported through the AcrB pore into the TolC tunnel (10.Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar). Indeed, AcrB structures with four structurally diverse substrates demonstrated that they bind to the large central cavity of AcrB (11.Yu E.W. McDermott G. Zgurskaya H.I. Nikaido H. Koshland Jr., D. Science. 2003; 300: 976-980Crossref PubMed Scopus (338) Google Scholar). An important question remains to be addressed is the role of AcrA in the efflux process. AcrA is essential for drug efflux, but how AcrA participates in this process is not fully understood. In its mature form, AcrA carries a diacylglycerol group and a palmitic acid chain linked to the N-terminal cysteine residue, which is believed to anchor the protein to the inner membrane. However, the lipid-deficient variant of AcrA carrying a His tag at the C-terminal is functional and has been used for biochemical studies (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). The secondary structure predictions of AcrA suggest that AcrA and its MFP homologs contain two regions of high coiled-coil probability of approximately equal length, flanked by two lipoyl/biotin-binding motifs that are likely β-strands (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). Although the high resolution structure is not available (14.Avila S. Misaghi S. Wilson K. Downing K.H. Zgurskaya H. Nikaido H. Nogales E. J. Struct. Biol. 2001; 136: 81-88Crossref PubMed Scopus (39) Google Scholar), AcrA was found to be a highly asymmetric molecule with an elongated shape of about 200 Å in length (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). Cross-linking experiments showed that AcrA forms a complex with AcrB (15.Zgurskaya H.I. Nikaido H. J. Bacteriol. 2000; 182: 4264-4267Crossref PubMed Scopus (141) Google Scholar), and may interact with TolC. Therefore, AcrA could provide a seamless link between AcrB and TolC. Alternatively, it may simply bring the outer and inner membranes closer for substrate transfer. A better knowledge of the structure and function of AcrA is essential for understanding the efflux process. In this study, we used a sensitive biophysical method, the site-directed spin labeling (SDSL), for studying the structure and dynamics of AcrA. SDSL utilizes site-directed mutagenesis to replace the residue of interest in a protein with a cysteine, which is then modified with a sulfhydryl-specific nitroxide to introduce the paramagnetic side chain (Fig. 1). Electron paramagnetic resonance (EPR) spectroscopic analysis of the spin label yields spectral characteristics that are dependent on the local environment, which in turn provide information on the structure and dynamics of the protein (16.Hubbell W.L. Gross A. Langen R. Lietzow M.A. Curr. Opin. Struct. Biol. 1998; 8: 649-656Crossref PubMed Scopus (500) Google Scholar, 17.Hubbell W.L. Cafiso D.S. Altenbach C. Nat. Struct. Biol. 2000; 7: 735-739Crossref PubMed Scopus (727) Google Scholar). Using this method, we demonstrate that AcrA is a dynamic protein that undergoes conformational rearrangements triggered by changes of pH. Such conformational changes may be important for the action of AcrA during the drug efflux process. Bacterial Strains, Plasmid Construction, and Growth Media—All E. coli strains were grown at 37 °C in LB broth. Antibiotics were added when required, to the following final concentrations: kanamycin, 34 μg/ml; ampicillin, 100 μg/ml; spectinomycin, 50 μg/ml; and chloramphenicol, 15 μg/ml. All plasmids were constructed by standard cloning techniques and propagated in the DH5α strain of E. coli. To construct pBP184, a 3557-bp HindIII-BamII fragment of pBP was ligated into a HindIII- and BamII-treated pACYC184 vector. Plasmid pBP184 expresses AcrB protein under the native acrAB promoter. The pUZ11 (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar) was used to construct all plasmids for the expression and purification of single cysteine containing AcrACys-6His. Plasmid pUZ11 encodes OmpA-AcrA-6His fusion protein, i.e. the signal sequence of OmpA protein was fused to the mature portion of AcrA (residues 26–394) lacking the AcrA signal peptide and the N-terminal cysteine modification site. The sixhistidine tag was at the C-terminal of AcrA. Unique cysteine residues were introduced into OmpA-AcrA-His6 using the QuickChange Site-directed Mutagenesis Kit (Stratagene). All final constructs were verified by DNA sequencing. Analysis of Function and Expression of AcrACys-6His Variants—The functional fitness of AcrACys-6His variants was evaluated by measuring minimal inhibitory concentrations (MICs) of different antibiotics. For this purpose, AcrACys-6His variants were transformed into AG100A (ΔacrAB::kan) strain carrying pBP184 plasmid (18.Okusu H. Ma D. Nikaido H. J. Bacteriol. 1996; 178: 306-308Crossref PubMed Scopus (623) Google Scholar). Then exponentially growing cultures (optical density at 600 nm of 1.0) were inoculated at a density of 104 cells per ml into LB medium in the presence of 2-fold-increasing concentrations of the drug under investigation. Cell growth was determined after overnight incubation at 37 °C. The levels of expression of AcrACys-6His were routinely monitored by Western immunoblotting analysis according to standard protocols using anti-AcrA antibody. E. coli cells were harvested by centrifugation, briefly sonicated, and solubilized by boiling in SDS sample buffer and subjected to SDS-polyacrylamide electrophoresis. In the case of the non-reducing SDS-PAGE, β-mercaptoethanol was omitted from the sample buffer. Protein Expression and Purification—The E. coli DH5α or AG100A strains containing plasmids with single cysteine substitution were cultured overnight. The overnight culture (5–10 ml) was inoculated into 500 ml of fresh LB medium with appropriate antibiotics and incubated at 37 °C until OD600 nm reached 0.5–0.7. Expression of protein was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside at 1 mm. After 3–4 h, cells were harvested by centrifugation, washed with 20 mm Tris-HCl, 0.2 m NaCl, pH 8.0, and subsequently disrupted by sonication. AcrA protein was purified from the cell lysates using His-Bind metal chelating resin (Novagen) according to the manufacturer's protocol with a slight modification (i.e. the concentration of imidazole in the Wash Buffer was 50 mm). To remove imidazole, protein was dialyzed against storage buffer (20 mm Tris-HCl, 0.2 m NaCl, 1 mm EDTA, pH 7.0) overnight. Purified protein is stable for at least 4 weeks at 4 °C. For prolonged storage, 10% glycerol was added, and protein was stored at –20 °C. The resulting protein was >95% pure as judged by SDS-PAGE and Coomassie Blue staining. Protein concentrations were determined at 280 nm using the molar extinction coefficient 17,210 cm–1m–1 or the BCA protein assay (Pierce Chemical Co.). Spin Labeling and EPR Spectroscopy—Thio-reducing agent DTT (4 mm) was added to the purified protein (∼0.5–2 mg/ml) in the storage buffer and incubated at 4 °C for 2–3 h. Samples were then dialyzed against the storage buffer overnight (2–3 buffer changes). Spin label MTSL, 1-oxy-2,2,5,5-tetramethylpyrrolinyl-3-methyl-methanethiosulfonate (Toronto Research Chemicals Inc., Toronto, Canada), was added at 10-fold molar excess with respect to the AcrA and incubated for 16 h at 4 °C. Excess free spin labels were removed by dialysis. For experiments at pH 5.0, another buffer, 25 mm MES, 0.2 m NaCl, 1 mm EDTA, pH 5.0, was used for dialysis. Proteins were concentrated by ultrafiltration and adjusted to 100–200 μm. For titration experiment, labeled AcrA in appropriate buffer was mixed with excess unlabeled AcrA (1:5 molar ratio) at protein concentration below 50 μm for overnight at 4 °C. The mixture was then concentrated and adjusted to 100–200 μm before acquiring EPR spectra. EPR spectra were obtained using a Bruker Elexsys E-500 spectrometer (Bruker, Rheinstetten, Germany). The conventional X-band spectra were obtained using 0.63 milliwatts microwave power and a field modulation of 100 kHz and 1.6 G modulation amplitude. Scan width was 100 G. EPR spectra were analyzed using the software Xport provided by the manufacturer (Bruker). Differential Scanning Calorimetry (DSC)—DSC experiments were performed on a MicroCal VP-DSC calorimeter (MicroCal, Northampton, MA). The protein concentration is at 1 mg/ml and the scan rate is 45 °C/hr. Data analysis was performed using Origin software provided by the manufacturer (MicroCal). Single Cysteine AcrA-6His Variants Are Functionally Active and Stable in Vivo—The native AcrA protein has a unique cysteine residue, Cys-25, which is the site for lipid modification (19.Ma D. Cook D.N. Alberti M. Pon N.G. Nikaido H. Hearst J.E. J. Bacteriol. 1993; 175: 6299-6313Crossref PubMed Google Scholar). However, genetic analysis suggested that neither lipid modification nor Cys-25 residue are essential for AcrA function (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). We have used the previously reported cysteine-less AcrAC25K variant with a hexa-histidine tag on the C terminus (AcrA-6His) as a background to construct 11 single cysteine AcrA (AcrACys-6His) mutants. Mutants were constructed by a PCR-based method and verified by sequencing. Previous sequence analysis predicted that AcrA has a highly hydrophilic central domain with a high propensity to form a trimeric coiled-coil and a C-terminal hydrophobic domain conserved among MFPs (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar, 20.Dinh T. Paulsen I.T. Saier M.H. J. Bacteriol. 1994; 176: 3825-3831Crossref PubMed Google Scholar). In addition, the central α-helical region of AcrA is bracketed by sequences homologous to lipoyl/biotin-binding domain (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). We introduced unique cysteine residues in all these putative domains of AcrA (Fig. 2). All 11 mutants could be expressed in E. coli to the similar level of wild-type AcrA (data not shown). We did not detect any visible degradation products, which could indicate protein instability induced by Cys substitutions. At most positions, cysteine substitutions were well tolerated by the presumed AcrA-AcrB-TolC complex, since plasmids expressing various AcrACys-6His proteins restored multidrug-resistant phenotype of cells lacking the chromosomal copy of acrA gene (Table I). In two instances, cells carrying AcrACys295 and AcrA-Cys390 exhibited only partial resistance to novobiocin and puromycin, compared with the wild-type AcrA. However, these mutants fully complemented resistance to other antimicrobials including erythromycin and tetracycline, and detergent SDS (Table I). Although these data do not address if initial rates of drug efflux are affected, they suggest that the mutant proteins are not grossly destabilized.Table IMinimal inhibitory concentrations (MIC) of various antimicrobial agents for E. coli cells expressing various AcrA mutant proteinsE. coli cells expressingMICErythromycinNovobiocinSDSPuromycinTetracyclineμg/mlAcrA6432>5000320.625AcrA A30C6416>5000320.625AcrA A39C6432>5000320.625AcrA A62C6432>5000320.625AcrA A103C6432>5000320.625AcrA A146C6416>5000320.625AcrA A172C6416>5000320.625AcrA A204C6416>5000320.625AcrA A242C6416>5000320.625AcrA A295C648>5000320.625AcrA A339C6432>5000320.625AcrA A390C648>5000160.625No AcrA (pUC18)424040.313 Open table in a new tab Spin Labeling and Stability of Labeled AcrACys-6His Variants—To gain insights into the molecular conformation and dynamics of AcrA in solution, ten AcrACys-6His variants were expressed, and purified by the standard methods, and subjected to spin labeling by a nitroxide reagent MTSL. All sites reacted readily with MTSL under the experimental conditions (i.e. without prior unfolding by denaturants) and gave strong EPR signals, suggesting that they are not buried within the structure and are accessible to MTSL, which is comparable in size to a tryptophan. As mentioned above, most cysteine substitutions did not alter the function of AcrA (Table I). We also examined if the presence of spin label MTSL affects AcrA stability by monitoring its thermal unfolding with DSC. All spin-labeled mutants examined display a single cooperative endotherm centered at a transition temperature of 50 °C, as illustrated by the representatives in Fig. 3. The corresponding cysteine mutants (unlabeled) and the parental AcrA protein were also analyzed. The DSC data were analyzed according to a two-state unfolding model, and the derived thermodynamic parameters are summarized in Table II. It is notable that changes of Tm and ΔH in the presence of spin label fall within the range of values found for substitutions at these sites by cysteine residues with the exception of 62R1, which is slightly more destabilized by the presence of MTSL. These results agree with previous findings from many studies that show MTSL, being relatively small, represents a minimal perturbation of the backbone fold, thermal stability, or function of a protein (21.Mchaourab H.S. Lietzow M.A. Hideg K. Hubbell W.L. Biochemistry. 1996; 35: 7692-7704Crossref PubMed Scopus (534) Google Scholar).Table IIThermodynamic properties of AcrA mutantsAcrA and mutantsTmΔH°Ckcal mol-1Parent AcrA52.0172Cys-3052.616530R151.9156Cys-3952.315539R152.8163Cys-6250.615362R146.3114Cys-10350.2179103R149.9159Cys-14650.3152146R149.2160Cys-17250.2146172R151.7168Cys-20452.8177204R152.8151Cys-24252.0170242R151.7176Cys-33952.2166339R150.0152Cys-36552.7150365R152.1173 Open table in a new tab Side Chain Mobility of Spin-labeled AcrA—Fig. 4 shows the EPR spectra of individual spin-labeled AcrA at pH 7.0. The 10 spectra are distinctive in terms of mobility, a qualitative descriptor of the dynamic modes of R1 in the protein. Two measurable parameters, the inverse central line width (ΔHo–1) and the spectral breadth that is represented by the separation of magnetic field between the two outermost peaks, have frequently been employed to determine the mobility of spin label, which reflects both the rate and amplitude of motion (17.Hubbell W.L. Cafiso D.S. Altenbach C. Nat. Struct. Biol. 2000; 7: 735-739Crossref PubMed Scopus (727) Google Scholar). Such mobility is determined by the immediate environment of a R1 residue that reflects its surrounding protein topography. Correlations between side chain mobility and the details of protein structure and dynamics have been studied extensively in several proteins including T4 lysozyme (T4L) (21.Mchaourab H.S. Lietzow M.A. Hideg K. Hubbell W.L. Biochemistry. 1996; 35: 7692-7704Crossref PubMed Scopus (534) Google Scholar), colicin E1 (22.Salwiäski L. Hubbell W.L. Protein Sci. 1999; 8: 562-572Crossref PubMed Scopus (28) Google Scholar), annexin XII (23.Langen R. Isas J.M. Hubbell W.L. Haigler H.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14060-14065Crossref PubMed Scopus (96) Google Scholar), and the T domain of diphtheria toxin (24.Oh K.J. Zhan H. Cui C. Hideg K. Collier R.J. Hubbell W.L. Science. 1996; 273: 810-812Crossref PubMed Scopus (114) Google Scholar). In general, R1 residues are highly immobilized at buried sites, immobilized or have complex multicomponent spectra at tertiary contact sites, and have relatively high mobility at exposed sites or in loop regions. The side chain mobility of AcrA, measured as ΔHo–1, was summarized in Table III. The spectra of 30R1 and 390R1 are dominated by sharp and narrow lineshapes, reflecting a high degree of motion. This suggests that the extremes of N and C termini of AcrA are largely unstructured, making little or no static tertiary contacts with the remainder of the protein. This is consistent with the notion that AcrA is in an extended conformation in solution and the N and C termini do not physically interact with each other but instead are located on two opposite poles of the protein molecule (12.Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar). In contrast, other R1 residues yield broadening spectra, which are characteristics of side chains within ordered structures or involving in tertiary interactions. The residues 103R1, 146R1, and 172R1 exhibit anisotropic motions that are typical of solvent-exposed, noninteracting, helix surface sites. The spectrum of 172R1 is complex, reflecting an anisotropic motion about an axis roughly parallel to the nitroxide 2pπ orbital. Nearly identical spectrum was described for residue 72R1 of T4L, which is located on the surface of a helix and experiences no interactions with nearby side chains (21.Mchaourab H.S. Lietzow M.A. Hideg K. Hubbell W.L. Biochemistry. 1996; 35: 7692-7704Crossref PubMed Scopus (534) Google Scholar). The cause for the constrained motion of 72R1 in T4L has been studied extensively and was recently shown to reflect the backbone motion (25.Columbus L. Hubbell W.L. Trends Biochem. Sci. 2002; 27: 288-295Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar). Consistent with the EPR spectra, residues 103R1, 146R1, and 172R1 are located at the predicted two α-helical regions with high probability of forming coiled-coil (Fig. 2).Table IIISide chain mobility of spin-labeled AcrA variantsAcrA variants(ΔHo)-1pH 7.0pH 5.0Gauss-130R10.67 ± 0.0030.67 ± 0.00339R10.42 ± 0.0040.38 ± 0.00362R10.42 ± 0.0030.29 ± 0.002103R10.48 ± 0.0020.40 ± 0.003146R10.31 ± 0.0020.25 ± 0.002172R10.37 ± 0.0030.32 ± 0.003204R10.40 ± 0.0020.41 ± 0.002242R10.26 ± 0.0030.24 ± 0.003339R10.30 ± 0.0030.29 ± 0.003390R10.67 ± 0.0030.67 ± 0.003 Open table in a new tab Residues 39R1, 62R1, and 204R1 have two spectral components, reflecting two resolved populations of different mobilities (α and β, Fig. 4). As described previously in many other systems, spin label attached to a single cysteine residues in protein often exhibits spectra composed of at least two motional components. Until recently, the structural basis remained unknown. The crystal structures of spin-labeled T4L proteins indicate that the multiple component spectra reflect bond rotational isomerization, which are modulated by interactions of the R1 side chain with neighbor side chains or backbone (26.Langen R. Oh K.J. Cascio D. Hubbell W.L. Biochemistry. 2000; 39: 8396-8405Crossref PubMed Scopus (227) Google Scholar). Thus, residues 39R1, 62R1, and 204R1 might be involved in tertiary contacts. Similarly, residues 242R1 and 339R1 may also have multiple dynamic components reflecting some degree of tertiary interactions (Fig. 4). Residues 62R1 and 204R1 are close to the predicted N- and C-lipoyl/biotin binding motifs, which are mostly β-strands (Fig. 2) (13.Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (143) Google Scholar). Residues 242R1 and 339R1 are close to the conserved C-terminal hydrophobic domain (20.Dinh T. Paulsen I.T. Saier M.H. J. Bacteriol. 1994; 176: 3825-3831Crossref PubMed Google Scholar). Although no secondary structures were predicted for regions of 39R1, 242R1, and 339R1, the EPR spectra indicate that these residues are located within ordered structures. In general, the EPR spectra are compatible with the predicted structure of AcrA based on sequence analyses. Conformational Rearrangements of AcrA Triggered by Changes of pH—The AcrAB-TolC system, like most bacterial efflux pumps, utilizes the proton motive force as the energy source for drug transport (6.Nikaido H. J. Bacteriol. 1996; 178: 5853-5859Crossref PubMed Scopus (873) Google Scholar). In E. coli cells, about half of the proton motive force across the cytoplasmic membrane comes from a proton gradient, with the cytoplasmic pH being higher than the external pH by about 1.7 pH unit (27.Nikaido H. Thanassi D.G. Antimicrob. Agents Chemother. 1993; 37: 1393-1399Crossref PubMed Scopus (228) Google Scholar). The in vitro reconstitution studies showed that AcrA greatly stimulates the transport activity of AcrB transporter by presumably promoting adhesion between two phospholipid bilayers (8.Zgurskaya H.I. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7190-7195Crossref PubMed Scopus (304) Google Scholar). In addition, AcrA alone could mediate hemifusion of lipid bilayer of membrane vesicles without intermixing the vesicle contents when there is a pH gradient across the vesicle (pH of vesicle interior is 7.0 and external 5.0) (8.Zgurskaya H.I. Nikaido H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 7190-7195Crossref PubMed Scopus (304) Google Scholar). Triggering of membrane fusion by lowering pH has been well established in the influenza viral membrane fusion protein, hemagglutinin (HA). HA is thought be in a metastable state at neutral pH and refolds into its lowest energy state by exposure to an acidic environment (28.Skehel J.J. Wiley D.C. Annu. Rev. Biochem. 2000; 69: 531-569Crossref PubMed Scopus (2240) Google Scholar). Therefore, we examine whether AcrA undergoes conformational transitions when the environment is acidified. Lowering the buffer pH from 7.0 to 5.0 caused changes of EPR spectra of residues 62R1, 103R1, 146R1, and 172R1, but little or no change of the others (i.e. 30R1, 39R1, 204R1, 242R1, 339R1, and 390R1, see Fig. 5A and Table III). Addition of MgCl2 (10 mm) did not cause further spectral changes (data not shown). The most pronounced spectral change induced by acidic pH was observed at residues 62R1 and 146R1. At pH 5.0, the spectrum of 62R1 became broadened and a strongly immobilized component appeared (see arrows in Fig. 5A), indicating that the local protein structure in the vicinity of 62R1 underwent a conformational change and consequently, residue 62R1 was in a more restricted environment. Residues 103R1 and 172R1 exhibited a similar conformational change, i.e. they became more restricted in mobility at pH 5.0. Comparing the EPR spectra of 146R1 at pH 7.0 and pH 5.0, the signal amplitude at pH 5.0 was decreased dramatically as the result of spectral broadening, and the baseline of the spectrum distorted, which are characteristics of spin-spin interaction that occurs when spin labels are at close proximity (<15 Å). Since all AcrA variants studied here are single labeled proteins, this result indicates that acidic pH induces oligomerization of AcrA, and that residue 146R1 is involved in intermolecular interaction. To further verify this, 146R1 mutant protein was titrated with unlabeled AcrA protein in five times excess and the spectra compared. Indeed, at pH 5.0, dilution of spin-labeled AcrA 146R1 mutant leads to spectral sharpening (Fig. 6), confirming that residue 146R1 is a tertiary contacting site among AcrA monomers. Interestingly, titration of 146R1 protein a
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