Small Multidrug Transporter EmrE 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.

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.

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.

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.

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.