Identification of Oligomerization and Drug-binding Domains of the Membrane Fusion Protein EmrA
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m209457200
ISSN1083-351X
AutoresM. Inês Borges‐Walmsley, Jeremy Beauchamp, Sharon M. Kelly, Kornelia Jumel, Denise Candlish, Stephen E. Harding, Nicholas C. Price, Adrian R. Walmsley,
Tópico(s)Antimicrobial Resistance in Staphylococcus
ResumoMany pathogenic Gram-negative bacteria possess tripartite transporters that catalyze drug extrusion across the inner and outer membranes, thereby conferring resistance. These transporters consist of inner (IMP) and outer (OMP) membrane proteins, which are coupled by a periplasmic membrane fusion (MFP) protein. However, it is not know whether the MFP translocates the drug between the membranes, by acting as a channel, or whether it brings the IMP and OMP together, facilitating drug transfer. The MFP EmrA has an elongated periplasmic domain, which binds transported drugs, and is anchored to the inner membrane by a single α-helix, which contains a leucine zipper dimerization domain. Consistent with CD and hydrodynamic analyses, the periplasmic domain is predicted to be composed of a β-sheet subdomain and an α-helical coiled-coil. We propose that EmrA forms a trimer in which the coiled-coils radiate across the periplasm, where they could sequester the OMP TolC. The "free" leucine zipper in the EmrA trimer might stabilize the interaction with the IMP EmrB, which also possesses leucine zipper motifs in the putative N- and C-terminal helices. The β-sheet subdomain of EmrA would sit at the membrane surface adjacent to the EmrB, from which it receives the transported drug, inducing a conformational change that triggers the interaction with the OMP. Many pathogenic Gram-negative bacteria possess tripartite transporters that catalyze drug extrusion across the inner and outer membranes, thereby conferring resistance. These transporters consist of inner (IMP) and outer (OMP) membrane proteins, which are coupled by a periplasmic membrane fusion (MFP) protein. However, it is not know whether the MFP translocates the drug between the membranes, by acting as a channel, or whether it brings the IMP and OMP together, facilitating drug transfer. The MFP EmrA has an elongated periplasmic domain, which binds transported drugs, and is anchored to the inner membrane by a single α-helix, which contains a leucine zipper dimerization domain. Consistent with CD and hydrodynamic analyses, the periplasmic domain is predicted to be composed of a β-sheet subdomain and an α-helical coiled-coil. We propose that EmrA forms a trimer in which the coiled-coils radiate across the periplasm, where they could sequester the OMP TolC. The "free" leucine zipper in the EmrA trimer might stabilize the interaction with the IMP EmrB, which also possesses leucine zipper motifs in the putative N- and C-terminal helices. The β-sheet subdomain of EmrA would sit at the membrane surface adjacent to the EmrB, from which it receives the transported drug, inducing a conformational change that triggers the interaction with the OMP. inner membrane membrane fusion protein inner membrane protein (a membrane transporter) carbenicillin 4-morpholineethanesulfonic acid minimum inhibitory concentration 4-morpholinepropanesulfonic acid nitrilotriacetic acid outer membrane outer membrane protein (an α/β-barrel protein channel) resistance-nodulation-cell division family of membrane transporters carbonyl cyanidep-trifluoromethoxyphenyl-hydrazone carbonyl cyanidem-chlorophenyl-hydrazone 2,4-dinitrophenol A major mechanism of resistance in pathogenic bacteria is the extrusion of antibiotics from the cell. Gram-negative bacteria possess tripartite transport systems for translocating drugs across both the inner membrane (IM)1 and the outer membrane (OM). This system consists of inner and outer membrane proteins, which translocate drugs across their respective membranes but are coupled by a periplasmic protein (1Borges-Walmsley M.I. Walmsley A.R. Trends Microbiol. 2001; 9: 71-79Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The periplasmic domain of this protein is apparently anchored to the IM via either a lipid moiety or an α-helix. There has been much speculation as to the functional role of this periplasmic protein, the delineation of which is crucial to understanding the mechanism of this type of transport system. One proposal is that it forms a channel between the membranes; but another suggests that it pulls the membranes together, allowing ligand transfer between the IMP and OMP (2Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (144) Google Scholar). Because of the latter hypothesis this periplasmic protein was originally termed a membrane fusion protein (MFP), but more recently the term dynamic adaptor has been adopted (3Andersen, C., Hughes, C., and Koronakis, V. EMBO Rep., 1, 313–318.Google Scholar). The structures of two of the components of such a tripartite complex, the OMP TolC (4Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar) and the IMP AcrB (5Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar), have recently been determined by x-ray crystallography (4Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar). Both TolC and AcrB crystallize as trimers. The three TolC molecules are structured into a 140-Å cylindrical channel with a 35-Å internal diameter. The OM end of the structure is open, providing solvent access, but the periplasmic end tapers to a virtual close. The structure can be divided into two major domains: an OM β-barrel and a periplasmic α-helical barrel. The β-barrel domain, which provides an essentially open channel through the OM, is composed of 12 β-strands, 4 donated by each TolC molecule, arranged into a right-twisted barrel. The α-helical domain is a 12-helix barrel, constructed from long (67 residues) and short (23 and 34 residues) helices, with pairs of the shorter helices stacked to produce pseudo-continuous helices. The α-helices are further arranged into coiled-coils, and the mixed α/β structure connecting the shorter helices forms a belt around the helical barrel. The α-helical barrel is about 100 Å long, which is close to the lower estimates of the depth of the periplasmic space at 130 Å, but some estimates put the depth of the periplasm at 250 Å and beyond the span of TolC (6Dubochet J. McDowall A.W. Menge B. Schmid E.N. Lickfeld K.G. J. Bacteriol. 1983; 155: 381-390Crossref PubMed Google Scholar, 7Graham L.L. Beveridge T.J. Naninga N. J. Bacteriol. 1991; 173: 1623-1633Crossref PubMed Google Scholar). The AcrB trimer, which has a jellyfish-like appearance, comprises a periplasmic headpiece with dimensions of 50 × >100 Å and a transmembrane domain with dimensions of 70 × >80 Å (5Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar). The headpiece, which is formed by protrusions between helices 1 and 2 and helices 7 and 8 of the transmembrane domain, is divided into two stacked parts, with the upper and lower parts 30 and 40 Å thick, respectively. Viewed from the side, the upper part has a trapezoidal appearance, 70 Å wide at the bottom and 40 Å at the top; whereas viewed from above, the upper part is open like a funnel, with an internal diameter of 30 Å. This funnel is connected by a pore, located between the headpieces of the three protomers, to a large central cavity at the interface of the headpiece and the transmembrane domains of the protomers. The three transmembrane domains, each of which is composed of 12 helices, are arranged into a ring with a 30-Å hole between them, which might be filled with phospholipids. It has been proposed that the upper headpiece interacts with TolC (5Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar), with six vertical hairpins from the AcrAB trimer contacting the six α-helix-turn-α-helix structures of the TolC trimer, to form a continuous path across the periplasmic space. If this is the case, it suggests a mechanism in which drugs transported through the transmembrane domains of AcrB are delivered to the central cavity created at the transmembrane domain headpiece interface, where they can be shuttled through the headpiece pore and funnel to TolC. Interestingly, MFPs are predicted to have a structure that resembles TolC; the N- and C termini of MFPs are proposed to fold into a flattened β-barrel, with the intervening residues arranged into two long helices, each of about 60 or more residues, which fold back on one another to form a coiled-coil (2Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (144) Google Scholar). Considering those MFPs that utilize an N-terminal α-helix to anchor them to the IM, this would position the β-barrel at the IM with the α-helices radiating out across the periplasm. Furthermore, the ability of MFPs to form stable trimers (8Thanabalu T. Koronakis E. Hughes C. Koronakis V. EMBO J. 1998; 17: 6487-6496Crossref PubMed Scopus (288) Google Scholar,9Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar) invites the suggestion that their role is to form a connecting channel between the IM translocase and TolC. The putative β-barrel of the MFP could act as the receiver domain for drugs released from the IM translocase, whereas the α-helices could transiently interact with TolC. A possible mechanism for this interaction is that the six α-helices of the MFP trimer form a cylinder that inserts into the closed end of TolC to open it. Considering however that both TolC and MFPs are highly elongated molecules (4Koronakis V. Sharff A. Koronakis E. Luisi B. Hughes C. Nature. 2000; 405: 914-919Crossref PubMed Scopus (870) Google Scholar, 9Zgurskaya H.I. Nikaido H. J. Mol. Biol. 1999; 285: 409-420Crossref PubMed Scopus (173) Google Scholar) capable of overlapping in the periplasmic space, a more likely mechanism is for the MFP to utilize its α-helices to "grab" the outer surface of TolC. There is a deep cleft within the headpiece of AcrB in which the MFP AcrA may lie, thereby positioning it to straddle both the periplasmic domains of AcrB and TolC (5Murakami S. Nakashima R. Yamashita E. Yamaguchi A. Nature. 2002; 419: 587-593Crossref PubMed Scopus (766) Google Scholar), and biochemical cross-linking studies have revealed that the MFP-TolC interaction is substrate-induced and transient (8Thanabalu T. Koronakis E. Hughes C. Koronakis V. EMBO J. 1998; 17: 6487-6496Crossref PubMed Scopus (288) Google Scholar). On the other hand, the β-domain contains a motif that resembles the lipoyl domain of enzymes involved in the transfer of a covalently attached lipoyl or biotinyl moiety between proteins (2Johnson J.M. Church G.M. J. Mol. Biol. 1999; 287: 695-715Crossref PubMed Scopus (144) Google Scholar). In such enzymes, this lipoyl domain is usually a flattened β-barrel. The formation of a similar domain would require the N- and C-terminal domains of the MFP to interact, which might provide a mechanism for bringing the two membranes together. However, the dimensions of AcrB and TolC are sufficient to indicate that they can contact one another across the periplasm, arguing against a role for the MFP in bringing the IM and OM together. This type of transport system is clearly of considerable scientific and medical interest, because our knowledge of them is rudimentary, and their study is likely to have medical benefits, because they confer drug resistance and only effect transport in bacteria. For this study, our aim was to characterize EmrA, the MFP of a multidrug transporter from Escherichia coli (10Lomovskaya O. Lewis K. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8938-8942Crossref PubMed Scopus (316) Google Scholar), as a structural and functional paradigm for the elucidation of the properties of a number of IMP-MFP-OMP transport systems and to address the central question of the role of the MFP in drug translocation. The EmrAB transporter is composed of EmrB, a putative 14-helix multidrug H+antiporter belonging to the major facilitator (MF) superfamily (11Pao S.S. Paulsen I.T. Saier M.H. Microbiol. Mol. Biol. Rev. 1998; 62: 1-34Crossref PubMed Google Scholar), and the MFP EmrA. EmrA is predicted to have a short N-terminal cytoplasmic domain, a single transmembrane helix, and a large periplasmic domain. The EmrAB proteins are thought to provide a continuous pathway across the bacterial membranes by operating in conjunction with TolC (1Borges-Walmsley M.I. Walmsley A.R. Trends Microbiol. 2001; 9: 71-79Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Underscoring the medical importance of this system, homologues of EmrA and B have been found in human pathogenic bacteria such as Vibrio cholerae (12Colmer J.A. Fralick J.A. Hamood A.N. Mol. Microbiol. 1998; 27: 63-72Crossref PubMed Scopus (85) Google Scholar),Neisseria gonorrhoeae (13Lee E.H. Shafer W.M. Mol. Microbiol. 1999; 33: 839-845Crossref PubMed Scopus (125) Google Scholar), Strenotrophomonas maltophilia (14Alonso A. Martinez J.L. Antimicrob. Agents Chemother. 2000; 44: 3079-3086Crossref PubMed Scopus (162) Google Scholar), and Campylobacter jejuni (15Lin J. Michel L.O. Zhang Q. Antimicrob. Agents Chemother. 2002; 46: 2124-2131Crossref PubMed Scopus (396) Google Scholar). Also the genome sequences of Bacillus subtilis, Haemophilus influenzae, Neisseria meningitidis, Bordatella pertussis, Rickettsia prowazeki, and Yersinia pestis indicate that they possess related systems. The pGEM-Teasy (Promega) plasmids bearing the emrA inserts were propagated in NovaBlue E. coli cells (Novagen). The pET21 (Novagen) plasmid constructs bearing emrA inserts were propagated inE. coli strain C41, a derivative of BL21 (16Miroux B. Walker J.E. J. Mol. Biol. 1996; 260: 289-298Crossref PubMed Scopus (1586) Google Scholar), and that bearing the hmrA insert was propagated in BL21* (Invitrogen). A ΔacrA strain of E. coli, termed N43 (17Ma D. Cook D.N. Alberti M. Pon N.G. Nikaido H. Hearst J.E. J Bacteriol. 1993; 175: 6299-6313Crossref PubMed Google Scholar), was transformed with E. coli emrAB, ΔNTemrAemrB (i.e. the construct expressed EmrA-(49–390)), and H. influenzae hmrAB pUC constructs and used for MIC measurements. Chromosomal DNA from E. coli strain DH5α was used as target DNA for amplification of emrA by PCR using the forward and reverse oligonucleotide primers 5′-GGATCCAGCGCAAATGCGGAGACTCA-3′ and 5′-CTCGAGGCCAGCGTTAGCTTTTACGAT-3′, respectively. We incorporated BamHI and XhoI restriction sites (underlined) into these primers to allow fragment ligation into pET21a to produce construct pET-EmrA-(1–390). Four truncated fragments of EmrA were generated by PCR, emrA-(15–390),emrA-(29–390), and emrA-(49–390), with the forward primers 5′-GGATCCAAGAGCGGCAAACGTAAG-3′, 5′-GGATCCC TCTTTATAATTATTGCCGT-3′ and 5′-GGATCCGAAGAAACCGATGACGCATACG-3′, respectively, in combination with the reverse primer used to cloneemrA-(1–390), whereas emrA-(1–56) was generated with the forward primer used to clone emrA-(1–390) and the reverse primer 5′-CTCGAGCGTATGCGTCATCGGTTTCTTC-3′. In each caseBamHI-XhoI restriction fragments were prepared and ligated into pET21a to produce constructs pET-EmrA-(15–390), pET-EmrA-(29–390), pET-EmrA-(49–390), and pET-EmrA-(1–56). Each pET construct was sequenced to ensure its integrity and that it was in-frame with the T7 and His6 tags. The pET constructs were used to transform E. coli strain C41, providing expression of the His6-tagged proteins. A single colony of C41/pET-EmrA was used to inoculate 5 ml of tryptone-yeast extract/50 μg ml−1 carbenicillin (CB), grown to saturation at 37 °C, and used to inoculate 0.5 liters of tryptone-yeast extract/50 μg ml−1 CB. Growth was continued at 37 °C until reaching anA600 of 0.4–0.5, at which point 1 mm isopropyl-1-thio-β-d-galactopyranoside/50 μg ml−1 CB was added, and the growth continued overnight at a reduced temperature of 23 °C. For most preparations 3 liters of cells (e.g. 6 × 0.5 liters) were cultivated. Cells were harvested at 8670 × g and washed with TNG buffer (50 mm Tris-HCl (pH 8.5), 150 mm NaCl, 10% glycerol). The cell pellet from each 0.5-liter culture was resuspended in 20 ml of TNG buffer, giving 100 ml for a 3-liter culture, to which was added 0.5 ml 10 mg ml−1 lysozyme, 0.1 ml 10 mg ml−1 DNase, and 1 protease inhibitor mixture tablet (complete, EDTA-freeTM, Roche Molecular Biochemicals). The cells were disrupted by passage through a cell disrupter (model Z-plus 1.1 kilowatts, Constant Systems) operated at 4 °C. Unbroken cells and cell debris were cleared from the supernatant at 39,000 × g (20 min, 4 °C), which was then spun at 194,000 × g (1.5 h, 4 °C), separating the soluble protein from the cell membrane pellet. EmrA-(49–390) was isolated from the supernatant, EmrA-(1–56), EmrA-(15–390), and EmrA-(1–390) from the membranes, and EmrA-(29–390) from both the membrane and soluble fractions. For purification of EmrA-(49–390), 6 ml of suspended Ni2+-NTA-agarose (Qiagen) was added to one-quarter of the supernatant and incubated for 1 h at 4 °C. A 1.5-cm-diameter Econo-columnTM (BioRad) was packed with the Ni2+-NTA-agarose to a final column volume of about 3 ml and washed successively with 10, 2.5, and 0.5 volumes of TNG buffer containing 10, 25, and 50 mm imidazole, respectively, before elution of the protein under gravity with 400 mmimidazole in 50 mm Tris-HCl (pH 8.5), 200 mmKCl (TK). Generally, 5 ml of protein at 15–20 mg ml−1 was eluted from the column, and then a further 5 ml of protein at 5 mg ml−1 was eluted, thus giving a yield of about 400 mg of protein from a 3-liter culture. The protein was dialyzed against TK to remove the imidazole. EmrA-(29–390) was obtained from the soluble fraction in an identical manner. For EmrA-(1–390), EmrA-(1–56), EmrA-(15–390), and EmrA-(29–390) (membrane fraction) purifications, each membrane pellet was solubilized by the addition of 1 volume of 1.5% dodecyl-β-d-maltoside in TKG buffer (TK plus 10% glycerol) and incubated on ice for 1 h, after which time 9 volumes of TKG was added to reduce the dodecyl-β-d-maltoside concentration to 0.15%, and solubilized proteins were separated from the membrane debris at 194,000 × g. To 100 ml of supernatant, 4 ml of suspended Ni2+-NTA-agarose/1 protease inhibitor tablet was added and incubated for 3 h at 4 °C. A 1-cm-diameter Econo-column was packed with the Ni2+-NTA-agarose to a final volume of about 2 ml and washed successively with 10, 2.5, and 2 volumes of TKGN (TKG plus 0.2%n-nonyl-β-d-maltoside) containing 10, 25, and 50 mm imidazole, respectively. 400 mmimidazole/TKGN was used to elute the protein under gravity, with the protein usually occurring in the second and third 1-ml aliquot at a concentration of 0.5–1 mg ml−1. The protein was dialyzed against TKGN to remove the imidazole. Each pET-EmrA construct was tested for expression in the cytoplasm, inner membrane, and periplasm by purification. Proteins were released from the periplasm by cold osmotic shock of the cells (18Hunt A.G. Hong J. Methods Enzymol. 1986; 125: 302-309Crossref PubMed Scopus (4) Google Scholar), and the protein extract was treated according to the procedure adopted for the purification of soluble EmrA proteins but maintaining the proteins in glycerol-containing buffers. Proteins were quickly frozen and stored at −80 °C. H. influenzaeRd, KW20, genomic DNA was obtained from the American Type Culture Collection and used to PCR clone the periplasmic domain of HmrA, the H. influenzaehomologue of EmrA, with the primers 5′-CACCATGTTTGAAGAAACAGAAGATGCTTATGTGG-3′ and 5′-ATGGCTGTTTTGCTGAATGATAGATTC-3′. The resulting product was cloned into the pET101/d-TOPO (Invitrogen) expression vector giving the pHmrA-6H plasmid, which was used to transform E. coli TOP10 cells, for overexpression of HmrA-(48–390). Automated DNA sequencing of the plasmid confirmed the sequence and translation frame as correct. pHmrA-6H was used to transform E. coliBL21* (Invitrogen), which was used for all subsequent protein production. BL21*/pHmrA-6H cells were grown at 37 °C in a 10-ml LB starter culture containing 100 μg/ml carbenicillin from a single colony picked from a fresh agar plate. When the cells were just visible, 1 ml of starter culture was used to inoculate 1 liter of LB containing 100 μg/ml CB, which was grown to an A600 of 0.4 at 37 °C with shaking at 200 rpm. Cells were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h at 25 °C and then chilled on ice for 1 h. The cells were harvested by centrifugation, resuspended in 50 mmHEPES (pH 8.0), 300 mm KCl, and disrupted with a Cell Disrupter (Constant Systems). The cell debris was removed by centrifugation and frozen in five 25-ml aliquots at −80 °C. HmrA-(48–390) was purified according to the same protocol adopted for EmrA-(49–390). The emrA and emrB genes were amplified by PCR using the forward and reverse primers 5′-GAATTCGAGCGCAAATGCGGAGACTC-3′ and 5′-GAAGCTTAGTGCGCACCTCCGCC-3′, respectively, to introduceEcoRI and HindIII sites (underlined) at the 5′ and 3′ ends of the amplified DNA, which was purified and ligated into pGEMT-Easy (Promega). The emrAB genes were rescued from pGEM-Teasy by restriction digest with EcoRI and HindIII, ligated intoEcoRI/HindIII-digested pUC to create pUC-EmrAB, and transformed into E. coli strain N43. Similarly, the pUC-EmrA-(49–390)B construct was made using the forward primer 5′-GAATTCGAAGAAACCGATGACGCATACG-3′, so that the expressed EmrA lacked the first 48 amino acids. The constructs were checked by automated DNA sequencing. The hmrA and hmrB genes were amplified by PCR using the forward and reverse primers 5′-GAATTCTGACGCAAATTGCAACT-3′ and 5′-GAAGCTTAATGCTGAGTACC AAA-3′, respectively. The PCR product was purified and ligated into pGEM-Teasy (Promega). The hmrAB genes were rescued from pGEM-Teasy by restriction digest with EcoRI, ligated intoEcoRI-digested, alkaline phosphatase-treated pUC to create pUC-HmrAB, and transformed into E. coli (NovoBlue, Invitrogen). Automated DNA sequencing was used to identify a plasmid in which the hmrAB genes were correctly orientated, which was then used to transform strain N43. MICs were measured according to the microdilution broth method established by the National Committee for Clinical Laboratory Standards (19National Committee for Clinical Laboratory Standards Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically.10. National Committee for Clinical Laboratory Standards, Villanova, PA1990Google Scholar). Briefly, a single colony was picked from an LB/ampicillin plate, used to inoculate 20 ml LB/ampicillin medium, and grown to an A600 of 0.1. Cells were transferred to a microtiter plate and mixed with serial dilutions of the drug to be tested. Bacterial growth was monitored after an 18-h incubation at 37 °C. Protein concentrations were determined by the BCA assay using a kit from Pierce with bovine serum albumin as standard. Proteins were separated by SDS-PAGE on 4–12% or 12% polyacrylamide gradient gels (NuPAGE gels and MES or MOPS buffer; Novex) and stained with Coomassie Gelcode BlueTM (Pierce). Protein samples for SDS-PAGE were mixed with the loading buffer at room temperature to avoid any potential problems due to protein aggregation, which can occur when samples are boiled. Native PAGE (7.5% (BioRad) polyacrylamide gels and Tris-glycine buffer (pH 7.5) run for 16 h at 50 v) was also used to separate proteins. EmrA proteins were subjected to gel chromatography on a Superdex 200 column run on an AKTA purifier (Amersham Biosciences) automated chromatography system. EmrA-(1–390) (1.3 mg ml−1 in TKGN) and EmrA-(49–390) (3.0 mg ml−1 in TKG) were applied to a Superdex 200 HR 10/30 column equilibrated with PNGL buffer (25 mm sodium phosphate (pH 8.0), 150 mm NaCl, 10% glycerol, 0.1% lauryldimethylamine-N-oxide) and eluted at a flow rate of 0.25 ml min−1. It was necessary to utilize PNGL as the equilibration buffer because the separation of the monomers and dimers of EmrA was inefficient when TKGN was used as the equilibration buffer, which however is better for storage of EmrA. Although the retention time for the EmrA-(49–390) monomer appeared to be less than for the EmrA-(1–390) dimer and monomer, possibly because the proteins adopt different conformations, the column resolution would be relatively poor in this range (e.g. between 45 and 90 kDa), and consequently there would be difficulties in comparing the elution profiles from different runs. Accordingly, we did not try to estimate the molecular masses of the proteins from the retention times. Moreover, the proteins are likely to elute as protein-detergent micelles with a greater molecular mass than the protein. EmrA-(1–390) monomers and dimers were separated by gel chromatography on a Superdex 200 column, resolved by 12% SDS-PAGE, transferred to polyvinylidene difluoride membrane and either immunoblotted with monoclonal anti-polyhistidine antibodies (Sigma) or N-terminal sequenced (Alta Bioscience, University of Birmingham, UK). The predicted amino acid sequence of the EmrA-(2–390)-(His)6 fusion protein (i.e. termed EmrA-(1–390)) was MASMTGGQQMGRDP-EmrA-(2–390)-LEHHHHHH, with residues 2–12 constituting an immunogenic T7 tag. N-terminal sequencing of the EmrA-(1–390) gave the first 11 and 10 residues of the T7 tag for the monomer and dimer, respectively. CD spectra were recorded on a Jasco J600 spectrometer at 20 °C using protein samples dialyzed into 50 mm Tris-acetic acid (pH 8.0), 200 mm NaF. The percentage of secondary structure was predicted from the CD spectra using the program SELCON (20Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32-44Crossref PubMed Scopus (946) Google Scholar). Fluorescence measurements were made in a Jasco FP750 fluorimeter at 20 °C. Tryptophan fluorescence was excited at 292.5 nm, and the emission wavelength was scanned between 300 and 400 nm. For titrations of EmrA-(49–390), KI was added from a 5 m stock solution to 2 ml of 1.25 μm protein in 50 mm Tris-HCl (pH 8.5), 200 mm NaCl. For KI titrations in the presence of drugs, the drug concentration was set to the maximum possible (e.g. 39 μm FCCP, 48 μm CCCP, 19 μm DNP, 20 μm nalidixic acid, and 53 μm chloramphenicol) that would not reduce the protein fluorescence by more than 50% because of its inner filter effect. In the case of nalidixic acid, the pH of the protein solutions was tested to ensure that the small volume of acid added had not perturbed the pH. The protein fluorescence and KI concentration were corrected for the dilution effect. HmrA-(48–390) was routinely used at a concentration of 6 μm because this gave a fluorescence equivalent to 1.25 μm EmrA-(49–390), but using the proteins at equivalent concentrations did not affect the titration curves. The FCCP titration curve for EmrA was fitted to an equation with hyperbolic and linear functions by nonlinear regression using the program SigmaPlot (Jandel Scientific): y = A − [(B × [FCCP]/Kd + [FCCP]) + (C × [FCCP])], where A is the value ofF0/F in the absence of FCCP,B is the total decrease in the value ofF0/F for concentrations of FCCP that tend toward saturating the hyperbolic component attributed to specific binding, Kd is the dissociation constant for the specific EmrA-(49–390)-FCCP complex, and C is the slope of the linear component attributed to nonspecific binding. Sedimentation velocity and sedimentation equilibrium measurements were made with an Optima XL-A analytical Ultracentrifuge (Beckman). Sedimentation was performed at 45,000 rpm in double sector cells at 20 °C, and the data were analyzed using DCDT+ software, version 1.12 (21Philo J.S. Anal. Biochem. 2000; 279: 151-163Crossref PubMed Scopus (239) Google Scholar). We measured the value of s for different protein concentrations in the range of 0.5–1 mg/ml in 40 mm potassium phosphate (pH 8.0), 400 mm KCl and extrapolated the data to zero protein concentration. Sedimentation equilibrium experiments were performed at 15,000, 18,000 and 20,000 rpm at 20 °C, and the data were analyzed using the manufacturer's software (Microcal Origin, version 4.1). The partial specific volume, ṽ, was calculated as 0.736 ml/g from the amino acid sequence of EmrA-(49–390) using SEDNTERP software (22Laue T.M. Shah B. Ridgeway T.M. Pelletier S. Harding S.E. Rowe A.J. Horton J. Analytical Ultracentrifugation in Biochemistry and Polymer Science. Royal Society of Chemistry, Cambridge, UK1992: 90-125Google Scholar); this value was used in all calculations. Dynamic light scattering experiments were performed on a Dynapro 801 (Protein Solutions Inc.) instrument at 20 °C. Samples were injected at a concentration of 1 mg ml−1. Data analysis was performed using the manufacturer's software. Residues 23–46 of EmrA were predicted, using the program TMHMM (23Krogh A. Larsson B. von Heijne G. Sonnhammer E.L.L. J. Mol. Biol. 2001; 305: 567-580Crossref PubMed Scopus (9290) Google Scholar), to form an α-helix that anchors the protein in the periplasm of E. coli. However, this prediction for EmrA, or for any other MFP, has not been tested experimentally. To map the domains of EmrA, a number ofemrA constructs were made in pET to overexpress the whole and truncated portions of EmrA (Fig. 1) with a His tag to aid in purification (Fig.2A). To determine the location of each protein, we attempted their purification from the periplasm milieu, obtained by osmotic shock of the cells, from the cytoplasmic milieu, obtained as the soluble fraction after cell disruption, and from inner membranes, prepared by detergent solubilization of membranes from disrupted cells. The EmrA-(1–390), EmrA-(15–390), and EmrA-(1–59) proteins were purified from cytoplasmic membranes, whereas EmrA-(49–390) was purified as a soluble protein from the cytoplasm. In contrast, EmrA-(29–390) was obtained from both membranes (about two-thirds of the total protein) and the cytoplasm (about one-third of the total protein). None of the proteins
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