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Identification of Oligomerization and Drug-binding Domains of the Membrane Fusion Protein EmrA*

  • M. Ines Borges-Walmsley
    Affiliations
    Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen's Campus, Stockton-on-Tees, TS17 6BH
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  • Jeremy Beauchamp
    Affiliations
    Division of Infection and Immunity and University of Glasgow, Glasgow G11 6NU

    Department of Chemistry, University of Glasgow, Glasgow G11 6NU
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  • Sharon M. Kelly
    Affiliations
    Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences and University of Glasgow, Glasgow G11 6NU
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  • Kornelia Jumel
    Affiliations
    National Center for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington LE12 5RD, United Kingdom
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  • Denise Candlish
    Affiliations
    Division of Infection and Immunity and University of Glasgow, Glasgow G11 6NU
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  • Stephen E. Harding
    Affiliations
    National Center for Macromolecular Hydrodynamics, School of Biosciences, University of Nottingham, Sutton Bonington LE12 5RD, United Kingdom
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  • Nicholas C. Price
    Affiliations
    Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences and University of Glasgow, Glasgow G11 6NU
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  • Adrian R. Walmsley
    Correspondence
    To whom correspondence should be addressed: Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen's Campus, Stockton-on-Tees TS17 6BH, United Kingdom. Tel.: 44-1642-333836; Fax: 44-1642-333817
    Affiliations
    Centre for Infectious Diseases, Wolfson Research Institute, University of Durham, Queen's Campus, Stockton-on-Tees, TS17 6BH
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  • Author Footnotes
    * This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BBSRC) and Wellcome Trust (to A. R. W.). The CD facility at the University of Glasgow and the National Center for Macromolecular Hydrodynamics at the University of Nottingham are both supported by the BBSRC and the Engineering and Physical Sciences Research Council (EPSRC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Open AccessPublished:December 13, 2002DOI:https://doi.org/10.1074/jbc.M209457200
      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.
      IM
      inner membrane
      MFP
      membrane fusion protein
      IMP
      inner membrane protein (a membrane transporter)
      CB
      carbenicillin
      MES
      4-morpholineethanesulfonic acid
      MIC
      minimum inhibitory concentration
      MOPS
      4-morpholinepropanesulfonic acid
      NTA
      nitrilotriacetic acid
      OM
      outer membrane
      OMP
      outer membrane protein (an α/β-barrel protein channel)
      RND
      resistance-nodulation-cell division family of membrane transporters
      FCCP
      carbonyl cyanidep-trifluoromethoxyphenyl-hydrazone
      CCCP
      carbonyl cyanidem-chlorophenyl-hydrazone
      DNP
      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 (
      • Borges-Walmsley M.I.
      • Walmsley A.R.
      ). 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 (
      • Johnson J.M.
      • Church G.M.
      ). 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 (

      Andersen, C., Hughes, C., and Koronakis, V. EMBO Rep., 1, 313–318.

      ).
      The structures of two of the components of such a tripartite complex, the OMP TolC (
      • Koronakis V.
      • Sharff A.
      • Koronakis E.
      • Luisi B.
      • Hughes C.
      ) and the IMP AcrB (
      • Murakami S.
      • Nakashima R.
      • Yamashita E.
      • Yamaguchi A.
      ), have recently been determined by x-ray crystallography (
      • Koronakis V.
      • Sharff A.
      • Koronakis E.
      • Luisi B.
      • Hughes C.
      ). 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 (
      • Dubochet J.
      • McDowall A.W.
      • Menge B.
      • Schmid E.N.
      • Lickfeld K.G.
      ,
      • Graham L.L.
      • Beveridge T.J.
      • Naninga N.
      ). 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 (
      • Murakami S.
      • Nakashima R.
      • Yamashita E.
      • Yamaguchi A.
      ). 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 (
      • Murakami S.
      • Nakashima R.
      • Yamashita E.
      • Yamaguchi A.
      ), 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 (
      • Johnson J.M.
      • Church G.M.
      ). 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 (
      • Thanabalu T.
      • Koronakis E.
      • Hughes C.
      • Koronakis V.
      ,
      • Zgurskaya H.I.
      • Nikaido H.
      ) 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 (
      • Koronakis V.
      • Sharff A.
      • Koronakis E.
      • Luisi B.
      • Hughes C.
      ,
      • Zgurskaya H.I.
      • Nikaido H.
      ) 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 (
      • Murakami S.
      • Nakashima R.
      • Yamashita E.
      • Yamaguchi A.
      ), and biochemical cross-linking studies have revealed that the MFP-TolC interaction is substrate-induced and transient (
      • Thanabalu T.
      • Koronakis E.
      • Hughes C.
      • Koronakis V.
      ). 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 (
      • Johnson J.M.
      • Church G.M.
      ). 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 (
      • Lomovskaya O.
      • Lewis K.
      ), 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 (
      • Pao S.S.
      • Paulsen I.T.
      • Saier M.H.
      ), 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 (
      • Borges-Walmsley M.I.
      • Walmsley A.R.
      ). Underscoring the medical importance of this system, homologues of EmrA and B have been found in human pathogenic bacteria such as Vibrio cholerae (
      • Colmer J.A.
      • Fralick J.A.
      • Hamood A.N.
      ),Neisseria gonorrhoeae (
      • Lee E.H.
      • Shafer W.M.
      ), Strenotrophomonas maltophilia (
      • Alonso A.
      • Martinez J.L.
      ), and Campylobacter jejuni (
      • Lin J.
      • Michel L.O.
      • Zhang Q.
      ). Also the genome sequences of Bacillus subtilis, Haemophilus influenzae, Neisseria meningitidis, Bordatella pertussis, Rickettsia prowazeki, and Yersinia pestis indicate that they possess related systems.

      DISCUSSION

      The aim of this study was to characterize EmrA, a membrane fusion protein from a tripartite multidrug extrusion system. By truncating the EmrA protein we were able to show that it is anchored to the inner membrane by residues 1–59, consistent with the proposal that residues 23–46 form a membrane-spanning α-helix, whereas residues 47–390 are arranged into a soluble periplasmic domain. Indeed, we found that EmrA-(15–390) was membrane-bound, whereas EmrA-(49–390) was soluble, indicating that residues 15–48, but not 1–15 or 49–390, are necessary for membrane insertion. Furthermore, EmrA-(29–390) is partially soluble, suggesting that the membrane-spanning helix starts between positions 15 and 29. We predict that the periplasmic domain of EmrA comprises a short β-sheet domain (residues 48–95), a large α-helical domain that is arranged into a coiled-coil (residues 96–213), and a large β-sheet domain (residues 214–374) with a short C-terminal helix (residues 375–387). This secondary structure prediction is reasonably consistent with a CD analysis of EmrA-(49–390). In common with the MFP AcrA (
      • Zgurskaya H.I.
      • Nikaido H.
      ) we found that the periplasmic domain of EmrA is highly elongated, with predicted dimensions of 27 × 2.3 nm. EmrA is predicted to have a stretch of 110 residues, with an α-helix structure, which is arranged into a coiled-coil. Assuming that there are 3.5 amino acids/helix turn, with a pitch of 0.51 nm/turn, we would expect this domain to have dimensions of 17 × 2 nm. Thus, both our CD and hydrodynamic data are reasonably consistent with the Jpred secondary structure prediction of a two-domain protein, e.g. a globular domain with a largely β-sheet structure capping an α-helical coiled-coil domain.
      Herein we provide evidence that membrane-bound EmrA forms dimers and trimers. What is the structural basis for the oligomerization of EmrA? EmrA is predicted to have an α-helical coiled-coil structure, which is a common motif in oligomeric proteins; the MULTICOIL program predicts that the coiled-coil domain will form dimers and trimers. Consistent with this prediction, we found that the soluble periplasmic domain of EmrA (e.g. EmrA-(49–390)) formed oligomers (at relatively high protein concentrations), but the dimers formed by membrane-bound whole EmrA (e.g. EmrA-(1–390)) were more stable. We note that both membrane-bound and soluble AcrA form dimers and trimers (
      • Balakrishnan L.
      • Hughes C.
      • Koronakis V.
      ). However, AcrA differs from EmrA in that it uses a lipid moiety to anchor it to the inner membrane, indicating that the oligomerization site for AcrA lies within the periplasmic domain. Another MFP, HlyD, was also shown to form trimers (
      • Thanabalu T.
      • Koronakis E.
      • Hughes C.
      • Koronakis V.
      ), suggesting that this is a common feature of MFPs. It seems most likely that a leucine zipper motif that runs through the N-terminal helix of EmrA acts as a dimerization domain, which stabilizes dimers of EmrA-(1–390) relative to those of EmrA-(49–390). Perhaps formation of the dimer provides a scaffold for trimerization, which results from interactions between the periplasmic domains of EmrA. In such a trimer the third leucine zipper motif would be “free.” Thus, it is of interest to note that both the putative N-terminal (e.g. Leu7, Ile14, Leu21, Leu28, and Val35, which span putative helix 1 between residues 13 and 38) and C-terminal helices (e.g. Leu473, Ile480, Ile487, and Leu494, which span putative helix 14 between residues 482 and 504) of EmrB contain leucine zipper motifs, which might interact with the free leucine zipper of the EmrA trimer to form a stable EmrA-EmrB complex. No leucine zipper motifs spanning the other putative helices of EmrB are apparent. Recent studies of HylD indicate that its cytosolic domain mediates transduction of the substrate binding signal to the periplasmic domain to trigger recruitment of TolC (
      • Sharff A.
      • Fanutti C.
      • Shi J.
      • Calladine C.
      • Luisi B.
      ). Thus, it is inviting to speculate that substrate binding to EmrB triggers communication between EmrB and EmrA via the leucine zippers, with signal propagation to the periplasmic domain of EmrA.
      EmrA and TolC are predicted to have similar tertiary and quaternary structures, an elongated α/β-barrel that forms trimers (
      • Andersen C.
      • Koronakis E.
      • Hughes C.
      • Koronakis V.
      ). This is suggestive of a related structure and function for these proteins, possibly with both acting to channel drugs across the periplasm. Indeed, the trimeric structure of EmrA is suggestive of the formation of a six-helix barrel, which could form a connecting channel with TolC. However, both TolC and EmrA are predicted to be sufficiently long to span most, if not all, of the periplasmic space, bringing into question how this interaction might be achieved if only the ends of the helical channels are involved. An alternative hypothesis might be one in which each α-helical coiled-coil of trimeric EmrA would act like “arms to grab” TolC, inducing the periplasmic end of TolC to adopt an open confirmation. Consistent with this hypothesis, recent studies indicate that a ring of aspartate residues (
      • Andersen C.
      • Koronakis E.
      • Bokma E.
      • Eswaran J.
      • Humphreys D.
      • Hughes C.
      • Koronakis V.
      ) and an intramolecular salt bridge (
      • Brooun A.
      • Tomashek J.J.
      • Lewis K.
      ) at the periplasmic end of TolC control the opening of the tunnel; their interaction with the MFP could be used to control the opening of TolC. However, there is evidence that the IMP and MFP, which are coupled, can work independently of the OMP (
      • Colmer J.A.
      • Fralick J.A.
      • Hamood A.N.
      ,
      • Chuanchuen R.
      • Narasaki C.T.
      • Schweizer H.P.
      ). It is possible that the MFP performs a role similar to that of TolC but channels the drugs to the outer membrane. Thermodynamically, the delivery of hydrophobic drugs to the outer membrane would be favorable.
      On the basis of the structure of the RND antiporter, AcrB, it has been suggested that the MFP AcrA binds to a large cleft in the periplasmic headpiece of AcrB (
      • Murakami S.
      • Nakashima R.
      • Yamashita E.
      • Yamaguchi A.
      ). In this position it would overlap the periplasmic domains of AcrB and TolC, where it could play a role in recruiting TolC. If this is the case, it brings into question the site of interaction of EmrA with EmrB, because EmrB is a major facilitator antiporter (
      • Borges-Walmsley M.I.
      • Walmsley A.R.
      ), which does not possess large periplasmic domains between helices 1 and 2 and helices 7 and 8. Perhaps the leucine zipper motif of EmrA is of importance in the EmrA-EmrB interaction, because it is noticeable that whereas EmrA is anchored by an α-helix, AcrA is anchored to the membrane by a lipid moiety. It has also been suggested that drugs can bind to the headpiece of AcrB and be channeled into the central pore region (
      • Murakami S.
      • Nakashima R.
      • Yamashita E.
      • Yamaguchi A.
      ). The headpiece contains vestibules that are open to the periplasm, which could be used to channel drugs to the pore at the center of the headpiece. Drugs delivered to the pore from the periplasm could then be delivered to TolC. Recent studies have shown that swapping the periplasmic domains of the two RND antiporters, AcrB and AcrD, which differ in their drug specificity, results in a change in the drug specificity of the chimeric proteins (
      • Elkins C.A.
      • Nikaido H.
      ), thus providing evidence that the periplasmic domains of AcrB are involved directly in drug binding. Herein we provide evidence that EmrA binds transported drugs, and it is tempting to speculate that EmrA may serve a similar role to that of the periplasmic domains of RND antiporters in drug binding and transfer to the OMP.
      A plausible model for the structure and function of the EmrB-EmrA-TolC tripartite transport system might be one in which EmrB and EmrA form a stable complex, possibly via their membrane-spanning leucine zipper motifs, positioning the β-sheet domain of EmrA above EmrB at the surface of the membrane, with the α-helices radiating out across the periplasm in position to contact TolC when triggered by the binding of drugs to the β-sheet domain of EmrA. The α-helical coiled-coils of EmrA would grab TolC so as to position the α-helical barrel of TolC above the β-sheet domain of EmrA. The drug could then be released from the β-sheet domain of EmrA, allowing it to diffuse into the channel formed by the TolC trimer. Although one can envisage the drug moving from a less hydrophobic site on EmrA/EmrB to a more hydrophobic site on TolC, it is puzzling as to how TolC rids itself of the drug. Clearly a detailed understanding of the function of EmrA will require knowledge of the three-dimensional structure, and toward this end we have recently succeeded in crystallizing EmrA.

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