Exploring the binding domain of EmrE, the smallest multidrug transporter

EmrE is a small multidrug transporter in Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons, thereby rendering cells resistant to these compounds. Biochemical experiments indicate that the basic functional unit of EmrE is a dimer where the common binding site for protons and substrate is formed by the interaction of an essential charged residue (Glu14) from both EmrE monomers. Previous studies implied that other residues in the vicinity of Glu14 are part of the binding domain. Alkylation of Cys replacements in the same transmembrane domain inhibits the activity of the protein and this inhibition is fully prevented by substrates of EmrE. To monitor directly the reaction we tested also the extent of modification using fluorescein-5-maleimide. While most residues are not accessible or only partially accessible, four, Y4C, I5C, L7C, and A10C, were modified at least 80%. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of two of these residues by up to 80%. To study other essential residues we generated functional hetero-oligomers and challenged them with various methane thiosulfonates. Taken together the findings imply the existence of a binding cavity accessible to alkylating reagents where at least three residues from TM1, Tyr40 from TM2, and Trp63 in TM3 are involved in substrate binding.     

A common binding site for substrates and protons in EmrE, an ion-coupled multidrug transporter

EmrE is an Escherichia coli 12-kDa multidrug transporter, which confers resistance to a variety of toxic cations by removing them from the cell interior in exchange with two protons. EmrE has only one membrane-embedded charged residue, Glu-14, that is conserved in more than 50 homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes.     

Precious things come in little packages

The 110-amino acid multidrug transporter from E. coli, EmrE, is a member of the family of MiniTexan or Smr drug transporters. EmrE can transport acriflavine, ethidium bromide, tetraphenylphosphonium (TPP+), benzalkonium and several other drugs with relatively high affinities. EmrE is an H+/drug antiporter, utilizing the proton electrochemical gradient generated across the bacterial cytoplasmic membrane by exchanging two protons with one substrate molecule. The EmrE multidrug transporter is unique in its small size and hydrophobic nature. Hydropathic analysis of the EmrE sequence predicts four alpha-helical transmembrane segments. This model is experimentally supported by FTIR studies that confirm the high alpha-helicity of the protein and by high-resolution heteronuclear NMR analysis of the protein structure. The TMS of EmrE are tightly packed in the membrane without any continuous aqueous domain, as was shown by Cysteine scanning experiments. These results suggest the existence of a hydrophobic pathway through which the substrates are translocated. EmrE is functional as a homo-oligomer as suggested by several lines of evidence, including co-reconstitution experiments of wild-type protein with inactive mutants in which negative dominance has been observed. EmrE has only one membrane embedded charged residue, Glu-14, that is conserved in more than fifty homologous proteins and it is a simple model system to study the role of carboxylic residues in ion-coupled transporters. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux and exchange reactions. We conclude that Glu-14 is an essential part of a binding site, common to substrates and protons. The occupancy of this site is mutually exclusive and provides the basis of the simplest coupling of two fluxes. Because of some of its properties and its size, EmrE provides a unique system to understand mechanisms of substrate recognition and translocation.     

A single carboxyl mutant of the multidrug transporter EmrE is fully functional

EmrE, a multidrug transporter from Escherichia coli removes toxic compounds from the cell in exchange with protons. Glu-14 is the only charged residue in the putative membrane domains and is fully conserved in more than 50 homologues of the protein. This residue was shown to be an essential part of the binding site, common to protons and substrate. EmrE bearing a single carboxylic residue, Glu-14, shows uptake and binding properties similar to those of the wild type. This suggests that a small protein bearing only 110 amino acids with a single carboxyl in position 14 is the most basic structure that shows ion-coupled transport activity. The role of Glu-14 in substrate binding was examined by using dicyclohexylcarbodiimide, a hydrophobic carbodiimide that is known to react with carboxyls. Tetraphenylphosphonium binding to both wild type and the single carboxyl mutant is inhibited by dicyclohexylcarbodiimide in a dose-dependent manner. Ethidium and other substrates of EmrE prevent this inhibition with an order of potency in accord with their apparent affinities. This suggests that dicyclohexylcarbodiimide binding is sterically prevented by the substrate, supporting the contention that Glu-14, the reactive residue, is part of the substrate-binding site.     

A model for coupling of H(+) and substrate fluxes based on "time-sharing" of a common binding site

Both prokaryotic and eukaryotic cells contain an array of membrane transport systems maintaining the cellular homeostasis. Some of them (primary pumps) derive energy from redox reactions, ATP hydrolysis, or light absorption, whereas others (ion-coupled transporters) utilize ion electrochemical gradients for active transport. Remarkable progress has been made in understanding the molecular mechanism of coupling in some of these systems. In many cases carboxylic residues are essential for either binding or coupling. Here we suggest a model for the molecular mechanism of coupling in EmrE, an Escherichia coli 12-kDa multidrug transporter. EmrE confers resistance to a variety of toxic cations by removing them from the cell interior in exchange for two protons. EmrE has only one membrane-embedded charged residue, Glu-14, which is conserved in more than 50 homologous proteins. We have used mutagenesis and chemical modification to show that Glu-14 is part of the substrate-binding site. Its role in proton binding and translocation was shown by a study of the effect of pH on ligand binding, uptake, efflux, and exchange reactions. The studies suggest that Glu-14 is an essential part of a binding site, which is common to substrates and protons. The occupancy of this site by H(+) and substrate is mutually exclusive and provides the basis of the simplest coupling for two fluxes.     

An amino acid cluster around the essential Glu-14 is part of the substrate- and proton-binding domain of EmrE,a multidrug transporter from Escherichia coli

EmrE is a small multidrug transporter (110 amino acids long) from Escherichia coli that extrudes various drugs in exchange with protons, thereby rendering bacteria resistant to these compounds. Glu-14 is the only charged membrane-embedded residue in EmrE and is evolutionarily highly conserved. This residue has an unusually high pK and is an essential part of the binding domain, shared by substrates and protons. The occupancy of the binding domain is mutually exclusive, and, as such, this provides the molecular basis for the coupling between substrate and proton fluxes. Systematic cysteine-scanning mutagenesis of the residues in the transmembrane segment (TM1), where Glu-14 is located, reveals an amino acid cluster on the same face of TM1 as Glu-14 that is part of the substrate- and proton-binding domain. Substitutions at most of these positions yielded either inactive mutants or mutants with modified affinity to substrates. Substitutions at the Ala-10 position, one helix turn away from Glu-14, yielded mutants with modified affinity to protons and thereby impaired in the coupling of substrate and proton fluxes. Taken as a whole, the results strongly support the concept of a common binding site for substrate and protons and stress the importance of one face of TM1 in substrate recognition, binding, and H(+)-coupled transport.     

An essential glutamyl residue in EmrE, a multidrug antiporter from Escherichia coli

EmrE is an Escherichia coli 12-kDa protein that confers resistance to toxic compounds, by actively removing them in exchange with protons. The protein includes eight charged residues. Seven of these residues are located in the hydrophilic loops and can be replaced with either Cys or another amino acid bearing the same charge, without impairing transport activity. Glu-14 is the only charged residue in the membrane domain and is conserved in all the proteins of the family. We show here that this residue is the site of action of dicyclohexylcarbodiimide, a carbodiimide known to act in hydrophobic environments. When Glu-14 was replaced with either Cys or Asp, resistance was abolished. Whereas the E14C mutant displays no transport activity, the E14D protein shows efflux and exchange at rates about 30-50% that of the wild type. The maximal DeltapH-driven uptake rate of E14D is only 10% that of the wild type. The mutant shows a different pH profile in all the transport modes. Our results support the notion that Glu-14 is an essential part of a binding domain shared by substrates and protons but mutually exclusive in time. This notion provides the molecular basis for the obligatory exchange catalyzed by EmrE.     

A membrane-embedded glutamate is required for ligand binding to the multidrug transporter EmrE

EmrE is an Escherichia coli multidrug transporter that confers resistance to a variety of toxins by removing them in exchange for hydrogen ions. The detergent-solubilized protein binds tetraphenylphosphonium (TPP(+)) with a K(D) of 10 nM. One mole of ligand is bound per approximately 3 mol of EmrE, suggesting that there is one binding site per trimer. The steep pH dependence of binding suggests that one or more residues, with an apparent pK of approximately 7.5, release protons prior to ligand binding. A conservative Asp replacement (E14D) at position 14 of the only membrane-embedded charged residue shows little transport activity, but binds TPP(+) at levels similar to those of the wild-type protein. The apparent pK of the Asp shifts to <5.0. The data are consistent with a mechanism requiring Glu14 for both substrate and proton recognition. We propose a model in which two of the three Glu14s in the postulated trimeric EmrE homooligomer deprotonate upon ligand binding. The ligand is released on the other face of the membrane after binding of protons to Glu14.     

Direct evidence for substrate-induced proton release in detergent-solubilized EmrE, a multidrug transporter

A novel approach to study coupling of substrate and ion fluxes is presented. EmrE is an H(+)-coupled multidrug transporter from Escherichia coli. Detergent-solubilized EmrE binds substrate with high affinity in a pH-dependent mode. Here we show, for the first time in an ion-coupled transporter, substrate-induced release of protons in a detergent-solubilized preparation. The direct measurements allow for an important quantitation of the phenomenon. Thus, stoichiometry of the release in the wild type and a mutant with a single carboxyl at position 14 is very similar and about 0.8 protons/monomer. The findings demonstrate that the only residue involved in proton release is a highly conserved membrane-embedded glutamate (Glu-14) and that all the Glu-14 residues in the EmrE functional oligomer participate in proton release. Furthermore, from the pH dependence of the release we determined the pK of Glu-14 as 8.5 and for an aspartate replacement at the same position as 6.7. The high pK of the carboxyl at position 14 is essential for coupling of fluxes of protons and substrates.     

Electron crystallography reveals plasticity within the drug binding site of the small multidrug transporter EmrE

EmrE is a Small Multidrug Resistance transporter (SMR) family member that mediates counter transport of protons and hydrophobic cationic drugs such as tetraphenylphosphonium (TPP⁺), ethidium, propidium and dequalinium. It is thought that the selectivity of the drug binding site in EmrE is defined by two negatively charged glutamate residues within a hydrophobic pocket formed from six of the α-helices, three from each monomer of the asymmetric EmrE homodimer. It is not apparent how such a binding pocket accommodates drugs of various sizes and shapes or whether the conformational changes that occur upon drug binding are identical for drugs of diverse chemical nature. Here, using electron cryomicroscopy of EmrE two-dimensional crystals we have determined projection structures of EmrE bound to three structurally different planar drugs, ethidium, propidium and dequalinium. Using image analysis and rigorous comparisons between these density maps and the density maps of the ligand-free and TPP⁺-bound forms of EmrE, we identify regions within the transporter that adapt differentially depending on the type of ligand bound. We show that all three planar drugs bind at the same pocket within the protein as TPP⁺. Furthermore, our analysis indicates that, while retaining the overall fold of the protein, binding of the planar drugs is accompanied by small rearrangements of the transmembrane domains that are different to those that occur when TPP⁺ binds. The regions in the EmrE dimer that are remodelled surround the drug binding site and include transmembrane domains from both monomers.     

Structure,Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.     

The key residue for substrate transport (Glu14) in the EmrE dimer is asymmetric

Transport proteins exhibiting broad substrate specificities are major determinants for the phenomenon of multidrug resistance. The Escherichia coli multidrug transporter EmrE, a 4-transmembrane, helical 12-kDa membrane protein, forms a functional dimer to transport a diverse array of aromatic, positively charged substrates in a proton/drug antiport fashion. Here, we report (13)C chemical shifts of the essential residue Glu(14) within the binding pocket. To ensure a native environment, EmrE was reconstituted into E. coli lipids. Experiments were carried out using one- and two-dimensional double quantum filtered (13)C solid state NMR. For an unambiguous assignment of Glu(14), an E25A mutation was introduced to create a single glutamate mutant. Glu(14) was (13)C-labeled using cell-free expression. Purity, labeling, homogeneity, and functionality were probed by mass spectrometry, NMR spectroscopy, freeze fracture electron microscopy, and transport assays. For Glu(14), two distinct sets of chemical shifts were observed that indicates structural asymmetry in the binding pocket of homodimeric EmrE. Upon addition of ethidium bromide, chemical shift changes and altered line shapes were observed, demonstrating substrate coordination by both Glu(14) in the dimer.     

In vitro monomer swapping in EmrE, a multidrug transporter from Escherichia coli, reveals that the oligomer is the functional unit

EmrE is a small multidrug transporter, 110 amino acids long that extrudes various drugs in exchange with protons, thereby rendering Escherichia coli cells resistant to these compounds. Negative dominance studies and radiolabeled substrate-binding studies suggested that EmrE functions as an oligomer. Projection structure of two-dimensional crystals of the protein revealed an asymmetric dimer. To identify the functional unit of EmrE, a novel approach was developed. In this method, quantitative monomer swapping is induced in detergent-solubilized EmrE by exposure to 80 degrees C, a treatment that does not impair transport activity. Oligomer formation is highly specific as judged by several criteria, among them the fact that (35)S-EmrE can be "pulled out" from a mixture prepared from generally labeled cells. Using this technique, we show that inactive mutant subunits are functionally complemented when mixed with wild type subunits. The hetero-oligomers thus formed display a decreased affinity to substrates. In addition, sulfhydryl reagents inhibit the above hetero-oligomer even though Cys residues are present only in the inactive monomer. It is concluded that, in EmrE, the oligomer is the functional unit.     

31P-CP-MAS NMR studies on TPP+ bound to the ion-coupled multidrug transport protein EmrE

The binding of tetraphenylphosphonium (TPP⁺) to EmrE, a membrane-bound, 110 residue Escherichia coli multidrug transport protein, has been observed by ³¹P cross-polarisation–magic-angle spinning nuclear magnetic resonance spectroscopy (CP–MAS NMR). EmrE has been reconstituted into dimyristoyl phosphatidylcholine bilayers. CP–MAS could selectively distinguish binding of TPP⁺ to EmrE in the fluid membrane. A population of bound ligand appears shifted 4 ppm to lower frequency compared to free ligand in solution, which suggests a rather direct and specific type of interaction of the ligand with the protein. This is also supported by the observed restricted motion of the bound ligand. The observation of another weakly bound substrate population arises from ligand binding to negatively charged residues in the protein loop regions.     

The projection structure of EmrE, a proton-linked multidrug transporter from Escherichia coli, at 7 A resolution

EmrE belongs to a family of eubacterial multidrug transporters that confer resistance to a wide variety of toxins by coupling the influx of protons to toxin extrusion. EmrE was purified and crystallized in two dimensions by reconstitution with dimyristoylphosphatidylcholine into lipid bilayers. Images of frozen hydrated crystals were collected by cryo-electron microscopy and a projection structure of EmrE was calculated to 7 A resolution. The projection map shows an asymmetric EmrE dimer with overall dimensions approximately 31 x 40 A, comprising an arc of highly tilted helices separating two helices nearly perpendicular to the membrane from another two helices, one tilted and the other nearly perpendicular. There is no obvious 2-fold symmetry axis perpendicular to the membrane within the dimer, suggesting that the monomers may have different structures in the functional unit.     

Conformational changes in the multidrug transporter EmrE associated with substrate binding

EmrE is a bacterial multidrug transporter of the small multidrug resistance family, which extrudes large hydrophobic cations such as tetraphenylphosphonium (TPP(+)) out of the cell by a proton antiport mechanism. Binding measurements were performed on purified EmrE solubilized in dodecylmaltoside to determine the stoichiometry of TPP(+) binding; the data showed that one TPP(+) molecule bound per EmrE dimer. Reconstitution of purified EmrE at low lipid:protein ratios in either the presence or the absence of TPP(+) produced well ordered two-dimensional crystals. Electron cryo-microscopy was used to collect images of frozen hydrated EmrE crystals and projection maps were determined by image processing to 7A resolution. An average native EmrE projection structure was calculated from the c222 and p222(1) crystals, which was subsequently subtracted from the average of two independent p2 projection maps of EmrE with TPP(+) bound. The interpretation of the difference density image most consistent with biochemical data suggested that TPP(+) bound at the monomer-monomer interface in the centre of the EmrE dimer, and resulted in the movement of at least one transmembrane alpha-helix.     

Substrate-induced tryptophan fluorescence changes in EmrE, the smallest ion-coupled multidrug transporter

Tryptophan residues may play several roles in integral membrane proteins including direct interaction with substrates. In this work we studied the contribution of tryptophan residues to substrate binding in EmrE, a small multidrug transporter of Escherichia coli that extrudes various positively charged drugs across the plasma membrane in exchange with protons. Each of the four tryptophan residues was replaced by site-directed mutagenesis. The only single substitutions that affected the protein's activity were those in position 63. While cysteine and tyrosine replacements yielded a completely inactive protein, the replacement of Trp63 with phenylalanine brought about a protein that, although it could not confer any resistance against the toxicants tested, could bind substrate with an affinity 2 orders of magnitude lower than that of the wild-type protein. Double or multiple cysteine replacements at the other positions generate proteins that are inactive in vivo but regain their activity upon solubilization and reconstitution. The findings suggest a possible role of the tryptophan residues in folding and/or insertion. Substrate binding to the wild-type protein and to a mutant with a single tryptophan residue in position 63 induced a very substantial fluorescence quenching that is not observed in inactive mutants or chemically modified protein. The reaction is dependent on the concentration of the substrate and saturates at a concentration of 2.57 microM with the protein concentration of 5 microM supporting the contention that the functional unit is a dimer. These findings strongly suggest the existence of an interaction between Trp63 and substrate, and the nature of this interaction can now be studied in more detail with the tools developed in this work.     

Comparison of three structures of the multidrug transporter EmrE

The small multidrug resistance proteins constitute a family of bacterial antiporters that confer multidrug resistance by H(+)-linked drug efflux across the bacterial cytoplasmic membrane. The structure of EmrE, the family archetype, has been determined by electron crystallography and shows that EmrE in the membrane is an asymmetric homodimer composed of a tightly packed bundle of eight alpha helices, six of which form the substrate-binding site, which has a single molecule of tetraphenylphosphonium at its centre. Two X-ray structures of EmrE have been determined; the first structure was of a non-native conformation of EmrE that formed a crystallographic tetramer, whereas EmrE in the second structure was an asymmetric dimer containing a single molecule of bound tetraphenylphosphonium. This recent EmrE structure bears a superficial resemblance to the electron crystallographic structure and the differences were ascribed to conformational changes. However, the biological relevance of these conformational differences is questionable.     

New insights into the structure and oligomeric state of the bacterial multidrug transporter EmrE: an unusual asymmetric homo-dimer

EmrE is a small multidrug transporter that contains 110 amino acid residues that form four transmembrane alpha-helices. The three-dimensional structure of EmrE has been determined from two-dimensional crystals by electron cryo-microscopy. EmrE is an asymmetric homo-dimer with one substrate molecule bound in a chamber accessible laterally from one leaflet of the lipid bilayer. Evidence from substrate binding analyses and analytical ultracentrifugation of detergent-solubilised EmrE shows that the minimum functional unit for substrate binding is a dimer. However, it is possible that EmrE exists as a tetramer in vivo and plausible models are suggested based upon analyses of two-dimensional crystals.