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.     

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.     

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.     

Anti-parallel membrane topology of a homo-dimeric multidrug transporter, EmrE

EmrE in Escherichia coli belongs to the small multidrug resistance (SMR) transporter family. It functions as a homo-dimer, but the orientation of the two monomers in the membrane (membrane topology) is under debate. We expressed various single-cysteine EmrE mutants in E. coli cells lacking a major efflux transporter. Efflux from cells expressing the P55C or T56C mutant was blocked by the external application of membrane-impermeable SH-reagents. This is difficult to explain by the parallel topology configuration, because Pro55 and Thr56 are considered to be located in the cytoplasm. From both the periplasm and the cytoplasm, biotin-PE-maleimide, a bulky membrane-impermeable SH-reagent, could access the cysteine residue at the 25th position in the presence of transport substrates and at the 108th position. These observations support the anti-parallel topology in the membrane.     

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.     

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.     

MAS solid-state NMR studies on the multidrug transporter EmrE

We study the uniformly ¹³C,¹⁵N isotopically enriched Escherichia coli multidrug resistance transporter EmrE using MAS solid-state NMR. Solid-state NMR can provide complementary structural information as the method allows studying membrane proteins in their native environment as no detergent is required for reconstitution. We compare the spectra obtained from wildtype EmrE to those obtained from the mutant EmrE-E14C. To resolve the critical amino acid E14, glutamic/aspartic acid selective experiments are carried out. These experiments allow to assign the chemical shift of the carboxylic carbon of E14. In addition, spectra are analyzed which are obtained in the presence and absence of the ligand TPP+.     

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.     

MAS solid-state NMR studies on the multidrug transporter EmrE

We study the uniformly 13C,15N isotopically enriched Escherichia coli multidrug resistance transporter EmrE using MAS solid-state NMR. Solid-state NMR can provide complementary structural information as the method allows studying membrane proteins in their native environment as no detergent is required for reconstitution. We compare the spectra obtained from wildtype EmrE to those obtained from the mutant EmrE-E14C. To resolve the critical amino acid E14, glutamic/aspartic acid selective experiments are carried out. These experiments allow to assign the chemical shift of the carboxylic carbon of E14. In addition, spectra are analyzed which are obtained in the presence and absence of the ligand TPP+.     

Role of sequence bias in the topology of the multidrug transporter EmrE

EmrE is the prototype of small multidrug resistance transporters and has emerged as a model of membrane protein evolution. Analysis of the distances separating symmetry-related site-specific spin labels, correlation of topological sequence bias to C-terminal orientation, to membrane insertion efficiency, and to resistance to ethidium bromide collectively demonstrate that EmrE monomers adopt a parallel topology in the functional dimer. We propose a coupled insertion and assembly model for EmrE in which the favorable energetics of the parallel dimer interface override topological constraints arising from weak asymmetry in positive charge distribution.     

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.     

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.     

Structure of the substrate binding pocket of the multidrug transporter EmrE: site-directed spin labeling of transmembrane segment 1

Site-directed spin labeling (SDSL) was used to explore the structural framework responsible for the obligatory drug-proton exchange in the Escherichia coli multidrug transporter, EmrE. For this purpose, a nitroxide scan was carried out along a stretch of 26 residues that include transmembrane segment 1 (TMS1). This segment has been implicated in the catalytic mechanism of EmrE due to the presence of the highly conserved glutamate 14, a residue absolutely required for ligand binding. Sequence-specific variation in the accessibilities of the introduced nitroxides to molecular oxygen reveals a transmembrane helical conformation along TMS1. One face of the helix is in contact with the hydrocarbon interior of the detergent micelle while the other face appears to be solvated by an aqueous environment, resulting in significant exposure of the nitroxides along this face to NiEDDA. TMS1 from two different subunits are in close proximity near a 2-fold axis of symmetry as revealed by the analysis of spin-spin interactions at sites 14 and 18. The limited extent of spin-spin interactions is consistent with a scissor-like packing of the two TMS1. This results in a V-shaped chamber which is in contact with the aqueous phase near the N-terminus. The spatial organization of TMS1, particularly the close proximity of E14, is consistent with a proposed mechanistic model of EmrE [Yerushalmi, H., and Schuldiner, S. (2000) Biochemistry 39, 14711-14719] where substrate extrusion is coupled to proton influx through electrostatic interactions and shifts of the glutamate 14 pK(a) during the cycle.     

The Escherichia coli multidrug transporter EmrE is a dimer in the detergent-solubilised state

EmrE is a multidrug transporter that utilises the proton gradient across bacterial cell membranes to pump hydrophobic cationic toxins out of the cell. The structure of EmrE is very unusual, because it is an asymmetric homodimer containing eight alpha-helices, six of which form the substrate-binding chamber and translocation pathway. Despite this structural information, the precise oligomeric order of EmrE in both the detergent-solubilised state and in vivo is unclear, although it must contain an even number of subunits to satisfy substrate-binding data. We have studied the oligomeric state of EmrE, purified in a functional form in dodecylmaltoside, by high-resolution size-exclusion chromatography (hrSEC) and by analytical ultracentrifugation. The data from equilibrium analytical ultracentrifugation were analysed using a measured density increment for the EmrE-lipid-detergent complex, which showed that the purified EmrE was predominantly a dimer. This value was consistent with the apparent mass for the EmrE-lipid-detergent complex (137 kDa) determined by hrSEC. EmrE was purified under different conditions using minimal concentrations of dodecylmaltoside, which would have maintained the structure of any putative higher oligomeric states: this EmrE preparation had an apparent mass of 206 kDa by hrSEC and equilibrium analytical ultracentrifugation showed unequivocally that EmrE was a dimer, although it was associated with a much larger mass of phospholipid. In addition, the effect of the substrate tetraphenylphosphonium on the oligomeric state was also analysed for both preparations of EmrE; velocity analytical ultracentrifugation showed that the substrate had no effect on the oligomeric state. Therefore, in the detergent dodecylmaltoside and under conditions where the protein is fully competent for substrate binding, EmrE is dimeric and there is no evidence from our data to suggest higher oligomeric states. These observations are discussed in relation to the recently published structures of EmrE from two- and three-dimensional crystals.     

Charge neutralization of the active site glutamates does not limit substrate binding and transport by small multidrug resistance transporter EmrE

EmrE, a small multidrug resistance transporter from Escherichia coli, confers broad-spectrum resistance to polyaromatic cations and quaternary ammonium compounds. Previous transport assays demonstrate that EmrE transports a +1 and a +2 substrate with the same stoichiometry of two protons:one cationic substrate. This suggests that EmrE substrate binding capacity is limited to neutralization of the two essential glutamates, E14_A and E14_B (one from each subunit in the antiparallel homodimer), in the primary binding site. Here, we explicitly test this hypothesis, since EmrE has repeatedly broken expectations for membrane protein structure and transport mechanism. We previously showed that EmrE can bind a +1 cationic substrate and proton simultaneously, with cationic substrate strongly associated with one E14 residue, whereas the other remains accessible to bind and transport a proton. Here, we demonstrate that EmrE can bind a +2 cation substrate and a proton simultaneously using NMR pH titrations of EmrE saturated with divalent substrates, for a net +1 charge in the transport pore. Furthermore, we find that EmrE can alternate access and transport a +2 substrate and proton at the same time. Together, these results lead us to conclude that E14 charge neutralization does not limit the binding and transport capacity of EmrE.     

Three-dimensional structure of the bacterial multidrug transporter EmrE shows it is an asymmetric homodimer

The small multidrug resistance family of transporters is widespread in bacteria and is responsible for bacterial resistance to toxic aromatic cations by proton-linked efflux. We have determined the three-dimensional (3D) structure of the Escherichia coli multidrug transporter EmrE by electron cryomicroscopy of 2D crystals, including data to 7.0 A resolution. The structure of EmrE consists of a bundle of eight transmembrane alpha-helices with one substrate molecule bound near the centre. The substrate binding chamber is formed from six helices and is accessible both from the aqueous phase and laterally from the lipid bilayer. The most remarkable feature of the structure of EmrE is that it is an asymmetric homodimer. The possible arrangement of the two polypeptides in the EmrE dimer is discussed based on the 3D density map.     

The membrane topology of EmrE - a small multidrug transporter from Escherichia coli

EmrE is a multidrug transporter from Escherichia coli that belongs to the Smr family of small multidrug transporters. The secondary structure of EmrE consists of a four helical bundle, as judged by different techniques. EmrE has been extensively characterized; nevertheless, the membrane topology of EmrE has not been determined yet. Previous work with a homologous Smr protein provided partial information of the membrane topology, however the location of the carboxy-terminus remained inconclusive. In this work we probed the membrane topology of EmrE, focusing on the carboxy-terminus of the protein, using two independent approaches. Our results support a secondary structure where the carboxy-terminus faces the cytoplasm, while the first loop faces the periplasm.     

EmrE, a multidrug transporter from Escherichia coli, transports monovalent and divalent substrates with the same stoichiometry

Multidrug transporters recognize and transport substrates with apparently little common structural features. At times these substrates are neutral, negatively, or positively charged, and only limited information is available as to how these proteins deal with the energetic consequences of transport of substrates with different charges. Multidrug transporters and drug-specific efflux systems are responsible for clinically significant resistance to chemotherapeutic agents in pathogenic bacteria, fungi, parasites, and human cancer cells. Understanding how these efflux systems handle different substrates may also have practical implications in the development of strategies to overcome the resistance mechanisms mediated by these proteins. Here, we compare transport of monovalent and divalent substrates by EmrE, a multidrug transporter from Escherichia coli, in intact cells and in proteoliposomes reconstituted with the purified protein. The results demonstrated that whereas the transport of monovalent substrates involves charge movement (i.e. electrogenic), the transport of divalent substrate does not (i.e. electroneutral). Together with previous results, these findings suggest that an EmrE dimer exchanges two protons per substrate molecule during each transport cycle. In intact cells, under conditions where the only driving force is the electrical potential, EmrE confers resistance to monovalent substrates but not to divalent ones. In the presence of proton gradients, resistance to both types of substrates is detected. The finding that under some conditions EmrE does not remove certain types of drugs points out the importance of an in-depth understanding of mechanisms of action of multidrug transporters to devise strategies for coping with the problem of multidrug resistance.     

Substrate binding to the multidrug transporter MepA

MepA is a multidrug transporter from Staphylococcus aureus that confers multidrug resistance through the efflux of a wide array of hydrophobic substrates. To evaluate the ability of MepA to recognize different substrates, the dissociation constants for interactions between MepA and three of its substrates (acriflavine (Acr), rhodamine 6G (R6G), and ethidium (Et)) were measured. Given that MepA is purified in the presence of detergents and that its substrates are hydrophobic, we examined the effect of the detergent concentration on the dissociation constant. We demonstrate that all three substrates interact directly with the detergent micelles. Additionally, we find the detergent effect on the K_D value to be highly substrate-dependent. The K_D value for R6G is greatly influenced by the detergent, whereas the K_D values for Acr and Et are only modestly affected. The effect of the inactive D183A mutant on binding was also evaluated. The D183A mutant shows lower affinity toward Acr and Et.     

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.