Small Multidrug Transporter Research Articles

BLaTM 2.0, a Genetic Tool Revealing Preferred Antiparallel Interaction of Transmembrane Helix 4 of the Dual-Topology Protein EmrE

Parallel and antiparallel transmembrane helix–helix interactions support the folding and non-covalent assembly of many integral membrane proteins. While several genetic tools are currently in use to study parallel transmembrane helix–helix interactions, antiparallel associations have been difficult to determine. Here, we present a novel genetic approach, termed BLaTM 2.0, which can be used in combination with the recently presented BLaTM 1.2 to compare the efficiency of antiparallel and parallel transmembrane domain (TMD) interactions in a natural membrane. In a practical application of the BLaTM system, we find that the antiparallel interaction of TMD4, the known dimerization domain of the dual-topology small multidrug transporter EmrE, is sequence-specific and much stronger than the parallel one. This suggests that TMD4 has evolved to favor the formation of dual-topology EmrE dimers over single-topology dimers.

Structural Dynamics of the Small Multidrug Transporter EMRE

The small multidrug transporter from E. coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient (1-2). While the principal elements of the antiport mechanism have been revealed (3), the structural record is limited to the crystal structure of the substrate-bound state (4). Here we utilize spin labeling and Q-band Double Electron Electron Resonance (DEER; 5) to uncover the conformational changes that are subsequent to protonation of critical acidic residues, a central and missing link in the structure/mechanism relationship. We find that protonation of E14 leads to extensive helix rotation and tilt in conjunction with repacking of loops, conformational changes which alter the orientation of side chains that coordinate substrate binding and modulate substrate access from the lipid bilayer. The model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers isoenergetic alternating access between inward- and outward facing conformations of the substrate-loaded transporter (6), thus enabling antiport without dissipating the proton gradient.(1) Schuldiner S (2009) Biochim Biophys Acta 1794(5): 748-762.(2) Amadi ST, Koteiche HA, Mishra S, Mchaourab HS (2010) J Biol Chem 285(34): 26710-26718.(3) Yerushalmi H, Schuldiner S (2000) FEBS Lett 476(1-2): 93-97.(4) Chen YJ, et al. (2007) Proc Natl Acad Sci USA 104(48): 18999-19004.(5) Jeschke G (2012) Annu Rev Phys Chem 63: 419-446.(6) Morrison EA, et al. (2012) Nature 481(7379): 45-50.

Protonation-dependent conformational dynamics of the multidrug transporter EmrE

The small multidrug transporter from Escherichia coli, EmrE, couples the energetically uphill extrusion of hydrophobic cations out of the cell to the transport of two protons down their electrochemical gradient. Although principal mechanistic elements of proton/substrate antiport have been described, the structural record is limited to the conformation of the substrate-bound state, which has been shown to undergo isoenergetic alternating access. A central but missing link in the structure/mechanism relationship is a description of the proton-bound state, which is an obligatory intermediate in the transport cycle. Here we report a systematic spin labeling and double electron electron resonance (DEER) study that uncovers the conformational changes of EmrE subsequent to protonation of critical acidic residues in the context of a global description of ligand-induced structural rearrangements. We find that protonation of E14 leads to extensive rotation and tilt of transmembrane helices 1-3 in conjunction with repacking of loops, conformational changes that alter the coordination of the bound substrate and modulate its access to the binding site from the lipid bilayer. The transport model that emerges from our data posits a proton-bound, but occluded, resting state. Substrate binding from the inner leaflet of the bilayer releases the protons and triggers alternating access between inward- and outward-facing conformations of the substrate-loaded transporter, thus enabling antiport without dissipation of the proton gradient.

Membrane Protein Misfolding Enforces the Positive-Inside Rule

Inner membrane proteins have long been known to follow the “positive-inside rule”, where cytoplasmic loops tend to have a greater number of cationic residues than periplasmic or extracellular loops. This effect can be seen dramatically in dual-topology proteins, such as the small multidrug transporter EmrE, where relatively balanced charge between the protein faces leads to mixed insertion in the membrane; some molecules are inserted with a cytoplasmic N-terminus, and some with a periplasmic. Addition or removal of positive charge can bias the orientation of the inserted protein. Surprisingly, this is true even if the mutation does not occur until after the synthesis of several transmembrane helices. Here, we examine how positive charges bring about orientation bias. We use a GFP-based assay to examine both the orientation of the protein as well as rates of misinsertion and degradation. We find a significant pool of degraded or misfolded protein to be present even with wild-type EmrE. In addition to the redistribution of well-folded protein, the amount, type, and orientation of degradation products changes dramatically upon the introduction of mutations that bias EmrE orientation. While early mutations, especially those surrounding the first transmembrane helix, appear capable of biasing the initial orientation, the effects of later mutations can be explained by altering the rate of misfolding events in accordance with the positive-inside rule. Positive charges thus appear to act as folding signals, enhancing proper folding and insertion when positioned in agreement with the positive-inside rule, and enhancing misfolding when placed in violation.

Specificity Determinants in Small Multidrug Transporters

Multiple-antibiotic resistance has become a major global public health concern, and to overcome this problem, it is necessary to understand the resistance mechanisms that allow survival of the microorganisms at the molecular level. One mechanism responsible for such resistance involves active removal of the antibiotic from the pathogen cell by MDTs (multidrug transporters). A prominent MDT feature is their high polyspecificity allowing for a single transporter to confer resistance against a range of drugs. Here we present the molecular mechanism underlying substrate recognition in EmrE, a small MDT from Escherichia coli. EmrE is known to have a substrate preference for aromatic, cationic compounds, such as methyl viologen (MV2+). In this work, we use a combined bioinformatic and biochemical approach to identify one of the major molecular determinants involved in MV2+ transport and resistance. Replacement of an Ala residue with Ser in weakly resistant SMRs from Bacillus pertussis and Mycobacterium tuberculosis enables them to provide robust resistance to MV2+ and to transport MV2+ and has negligible effects on the interaction with other substrates. This shows that the residue identified herein is uniquely positioned in the binding site so as to be exclusively involved in the mediating of MV2+ transport and resistance, both in EmrE and in other homologues. This work provides clues toward uncovering how specificity is achieved within the binding pocket of a polyspecific transporter that may open new possibilities as to how these transporters can be manipulated to bind a designed set of drugs.

A Small Multidrug Resistance-like Transporter Involved in the Arabinosylation of Arabinogalactan and Lipoarabinomannan in Mycobacteria

The biosynthesis of the major cell envelope glycoconjugates of Mycobacterium tuberculosis is topologically split across the plasma membrane, yet nothing is known of the transporters required for the translocation of lipid-linked sugar donors and oligosaccharide intermediates from the cytoplasmic to the periplasmic side of the membrane in mycobacteria. One of the mechanisms used by prokaryotes to translocate lipid-linked phosphate sugars across the plasma membrane relies on translocases that share resemblance with small multidrug resistance transporters. The presence of an small multidrug resistance-like gene, Rv3789, located immediately upstream from dprE1/dprE2 responsible for the formation of decaprenyl-monophosphoryl-β-D-arabinose (DPA) in the genome of M. tuberculosis led us to investigate its potential involvement in the formation of the major arabinosylated glycopolymers, lipoarabinomannan (LAM) and arabinogalactan (AG). Disruption of the ortholog of Rv3789 in Mycobacterium smegmatis resulted in a reduction of the arabinose content of both AG and LAM that accompanied the accumulation of DPA in the mutant cells. Interestingly, AG and LAM synthesis was restored in the mutant not only upon expression of Rv3789 but also upon that of the undecaprenyl phosphate aminoarabinose flippase arnE/F genes from Escherichia coli. A bacterial two-hybrid system further indicated that Rv3789 interacts in vivo with the galactosyltransferase that initiates the elongation of the galactan domain of AG. Biochemical and genetic evidence is thus consistent with Rv3789 belonging to an AG biosynthetic complex, where its role is to reorient DPA to the periplasm, allowing this arabinose donor to then be used in the buildup of the arabinan domains of AG and LAM.

Characterization of co-translationally formed nanodisc complexes with small multidrug transporters, proteorhodopsin and with the E. coli MraY translocase

Nanodiscs (NDs) enable the analysis of membrane proteins (MP) in natural lipid bilayer environments. In combination with cell-free (CF) expression, they could be used for the co-translational insertion of MPs into defined membranes. This new approach allows the characterization of MPs without detergent contact and it could help to identify effects of particular lipids on catalytic activities. Association of MPs with different ND types, quality of the resulting MP/ND complexes as well as optimization parameters are still poorly analyzed. This study describes procedures to systematically improve CF expression protocols for the production of high quality MP/ND complexes. In order to reveal target dependent variations, the co-translational ND complex formation with the bacterial proton pump proteorhodopsin (PR), with the small multidrug resistance transporters SugE and EmrE, as well as with the Escherichia coli MraY translocase was studied. Parameters which modulate the efficiency of MP/ND complex formation have been identified and in particular effects of different lipid compositions of the ND membranes have been analyzed. Recorded force distance pattern as well as characteristic photocycle dynamics indicated the integration of functionally folded PR into NDs. Efficient complex formation of the E. coli MraY translocase was dependent on the ND size and on the lipid composition of the ND membranes. Active MraY protein could only be obtained with ND containing anionic lipids, thus providing new details for the in vitro analysis of this pharmaceutically important protein.

Undecided membrane proteins insert in random topologies. Up, down and sideways: it does not really matter

It is usually assumed that to ensure proper function, membrane proteins must be inserted in a unique topology. However, a number of dimeric small multidrug transporters can function in the membrane in various topologies. Thus, the dimers can be a random mixture of NiCi (N and C termini facing the cell cytoplasm) and NoCo (N and C termini facing the outside) orientation. In addition, the dimer functions whether the two protomers are parallel (N and C termini of both protomers on the same side of the membrane) or antiparallel (N and C termini of each protomer on opposite sides of the membrane). This unique phenomenon provides strong support for a simple mechanism of transport where the directionality is determined solely by the driving force.

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.

Topologically Random Insertion of EmrE Supports a Pathway for Evolution of Inverted Repeats in Ion-coupled Transporters

Inverted repeats in ion-coupled transporters have evolved independently in many unrelated families. It has been suggested that this inverted symmetry is an essential element of the mechanism that allows for the conformational transitions in transporters. We show here that small multidrug transporters offer a model for the evolution of such repeats. This family includes both homodimers and closely related heterodimers. In the former, the topology determinants, evidently identical in each protomer, are weak, and we show that for EmrE, an homodimer from Escherichia coli, the insertion into the membrane is random, and dimers are functional whether they insert into the cytoplasmic membrane with the N- and C-terminal domains facing the inside or the outside of the cell. Also, mutants designed to insert with biased topology are functional regardless of the topology. In the case of EbrAB, a heterodimer homologue supposed to interact antiparallel, we show that one of the subunits, EbrB, can also function as a homodimer, most likely in a parallel mode. In addition, the EmrE homodimer can be forced to an antiparallel topology by fusion of an additional transmembrane segment. The simplicity of the mechanism of coupling ion and substrate transport and the few requirements for substrate recognition provide the robustness necessary to tolerate such a unique and unprecedented ambiguity in the interaction of the subunits and in the dimer topology relative to the membrane. The results suggest that the small multidrug transporters are at an evolutionary junction and provide a model for the evolution of structure of transport proteins.

In vitro Unfolding and Refolding of the Small Multidrug Transporter EmrE

The composition of the lipid bilayer is increasingly being recognised as important for the regulation of integral membrane protein folding and function, both in vivo and in vitro. The folding of only a few membrane proteins, however, has been characterised in different lipid environments. We have refolded the small multidrug transporter EmrE in vitro from a denatured state to a functional protein and monitored the influence of lipids on the folding process. EmrE is part of a multidrug resistance protein family that is highly conserved amongst bacteria and is responsible for bacterial resistance to toxic substances. We find that the secondary structure of EmrE is very stable and only small amounts are denatured even in the presence of unusually high denaturant concentrations involving a combination of 10 M urea and 5% SDS. Substrate binding by EmrE is recovered after refolding this denatured protein into dodecylmaltoside detergent micelles or into lipid vesicles. The yield of refolded EmrE decreases with lipid bilayer compositional changes that increase the lateral chain pressure within the bilayer, whilst conversely, the apparent rate of folding seems to increase. These results add further weight to the hypothesis that an increased lateral chain pressure hinders protein insertion across the bilayer. Once the protein is inserted, however, the greater pressure on the transmembrane helices accelerates correct packing and final folding. This work augments the relatively small number of biophysical folding studies in vitro on helical membrane proteins.

Identification of a Glycine Motif Required for Packing in EmrE, a Multidrug Transporter from Escherichia coli

Glycine residues may play functional and structural roles in membrane proteins. In this work we studied the role of glycine residues in EmrE, a small multidrug transporter from Escherichia coli. EmrE extrudes various drugs across the plasma membrane in exchange with protons and, as a result, confers resistance against their toxic effects. Each of 12 glycine residues was replaced by site-directed mutagenesis. Four of the 12 glycine residues in EmrE are evolutionary conserved within the small multidrug resistance family of multidrug transporters. Our analysis reveals that only two (Gly-67 and Gly-97) of these four highly conserved residues are essential for transporter activity. Moreover, two glycine positions that are less conserved, Gly-17 and Gly-90, demonstrate also a nil phenotype when substituted. Our present results identifying Gly-17 and Gly-67 as irreplaceable reinforce the importance of previously defined functional clusters. Two essential glycine residues, Gly-90 and Gly-97, form a protein motif in which glycine residues are separated by six other residues (GG7). Upon substitution of glycine in these positions, the protein ability to form dimers is impaired as evaluated by cross-linking and pull-down experiments.

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.

Cell‐free expression as an emerging technique for the large scale production of integral membrane protein

Membrane proteins are highly underrepresented in structural data banks due to tremendous difficulties that occur upon approaching their structural analysis. Inefficient sample preparation from conventional cellular expression systems is in many cases the first major bottleneck. Preparative scale cell-free expression has now become an emerging alternative tool for the high level production of integral membrane proteins. Many toxic effects attributed to the overproduction of recombinant proteins are eliminated by cell-free expression as viable host cells are no longer required. A unique characteristic is the open nature of cell-free systems that offers a variety of options to manipulate the reaction conditions in order to protect or to stabilize the synthesized recombinant proteins. Detergents or lipids can easily be supplemented and membrane proteins can therefore be synthesized directly into a defined hydrophobic environment of choice that permits solubility and allows the functional folding of the proteins. Alternatively, cell-free produced precipitates of membrane proteins can efficiently be solubilized in mild detergents after expression. Highly valuable for structural approaches is the fast and efficient cell-free production of uniformly or specifically labeled proteins. A considerable number of membrane proteins from diverse families like prokaryotic small multidrug transporters or eukaryotic G-protein coupled receptors have been produced in cell-free systems in high amounts and in functionally active forms. We will give an overview about the current state of the art of this new approach with special emphasis on technical aspects as well as on the functional and structural characterization of cell-free produced membrane proteins.

Identification of Tyrosine Residues Critical for the Function of an Ion-coupled Multidrug Transporter

Aromatic residues may play several roles in integral membrane proteins, including direct interaction with substrates. In this work, we studied the contribution of tyrosine residues to the activity of EmrE, a small multidrug transporter from Escherichia coli that extrudes various drugs across the plasma membrane in exchange with protons. Each of five tyrosine residues was replaced by site-directed mutagenesis. Two of these residues, Tyr-40 and Tyr-60, can be partially replaced with hydroxyamino acids, but in the case of Tyr-40, replacement with either Ser or Thr generates a protein with modified substrate specificity. Replacement of Tyr-4 with either Trp or Phe generates a functional transporter. A Cys replacement at this position generates an uncoupled protein; it binds substrate and protons and transports the substrate downhill but is impaired in uphill substrate transport in the presence of a proton gradient. The role of these residues is discussed in the context of the published structures of EmrE.

Microcalorimetry of EmrE, a Multi‐Drug Transporter.

The efflux of structurally diverse toxic substrates by multi-drug transporters is the cause of significant resistance of pathogenic bacteria to a number of therapeutic compounds. EmrE is a small multi-drug transporter from Escherichia coli that utilises the proton gradient to extrude a number of hydrophobic cations from the cell, thus resulting in resistance to toxic compounds. Interactions between EmrE and drug molecules can be determined by microcalorimetry, which also allows non-invasive determination of absolute thermodynamic quantities. These interactions can also be measured and quantified in lipid environments that are not amenable to other biophysical techniques. We have investigated the thermodynamic parameters of binding of the substrate tetraphenylphosphonium (TPP+) to EmrE, reconstituted in various detergent and lipid environments, by Isothermal Titration Calorimetry. The Stoiciometry and binding affinity is in agreement with previous work, with a stoichiometry of 1 molecule of ligand to each EmrE dimer.

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.

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.

Exploring the Role of a Unique Carboxyl Residue in EmrE by Mass Spectrometry

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 (Glu-14) from both EmrE monomers. Carbodiimide modification of EmrE has been studied using functional assays, and the evidence suggests that Glu-14 is the target of the reaction. Here we exploited electrospray ionization mass spectrometry to directly monitor the reaction with each monomer rather than following inactivation of the functional unit. A cyanogen bromide peptide containing Glu-14 allows the extent of modification by the carboxyl-specific modification reagent diisopropylcarbodiimide (DiPC) to be monitored and reveals that peptide 2NPYIYLGGAILAEVIGTTLM(21) is approximately 80% modified in a time-dependent fashion, indicating that each Glu-14 residue in the oligomer is accessible to DiPC. Furthermore, preincubation with tetraphenylphosphonium reduces the reaction of Glu-14 with DiPC by up to 80%. Taken together with other biochemical data, the findings support a "time sharing" mechanism in which both Glu-14 residues in a dimer are involved in tetraphenylphosphonium and H(+) binding.

The Reconstitution and Activity of the Small Multidrug Transporter EmrE is Modulated by Non-bilayer Lipid Composition

The ability of multidrug transport proteins within biological membranes to recognise a diverse array of substrates is a fundamental aspect of antibiotic resistance. Detailed information on the mechanisms of recognition and transport can be provided only by in vitro studies in reconstituted bilayer systems. We describe the controlled, efficient reconstitution of the small multidrug transporter EmrE in a simple model membrane and investigate the effect of non-bilayer lipids on this process. Transport activity is impaired, in line with an increase in the lateral pressure within the bilayer. We demonstrate the potential of this lateral pressure modulation method as a general approach to the folding and assembly of membrane proteins in vitro, by recovering functional transporter from a partly denatured state. Our results highlight the importance of optimising reconstitution procedures and bilayer lipid composition in studies of membrane transporters. This is particularly pertinent for multidrug proteins, and we show that the use of a sub-optimal lipid bilayer environment or reconstitution method could lead to incorrect information on protein activity.