SmFRET Probing Reveals Substrate-Dependent Conformational Dynamics of E. coli Multidrug MdfA.
ABSTRACT: Bacterial multidrug-resistance transporters of the major facilitator superfamily are distinguished by their extraordinary ability to bind structurally diverse substrates, thus serving as a highly efficient tool to protect cells from multiple toxic substances present in their environment, including antibiotic drugs. However, details of the dynamic conformational changes of the transport cycle involved remain to be elucidated. Here, we used the single-molecule fluorescence resonance energy transfer technique to investigate the conformational behavior of the Escherichia coli multidrug transporter MdfA under conditions of different substrates, pH, and alkali metal ions. Our data show that different substrates exhibit distinct effects on both the conformational distribution and transition rate between two major conformations. Although the cationic substrate tetraphenylphosphonium favors the outward-facing conformation, it has less effect on the transition rate. In contrast, binding of the electroneutral substrate chloramphenicol tends to stabilize the inward-facing conformation and decreases the transition rate. Therefore, our study supports the notion that the MdfA transporter uses distinct mechanisms to transport electroneutral and cationic substrates.
Project description:Multidrug transporters are integral membrane proteins that use cellular energy to actively extrude antibiotics and other toxic compounds from cells. The multidrug/proton antiporter MdfA from Escherichia coli exchanges monovalent cationic substrates for protons with a stoichiometry of 1, meaning that it translocates only one proton per antiport cycle. This may explain why transport of divalent cationic drugs by MdfA is energetically unfavorable. Remarkably, however, we show that MdfA can be easily converted into a divalent cationic drug/? 2 proton-antiporter, either by random mutagenesis or by rational design. The results suggest that exchange of divalent cationi c drugs with two (or more) protons requires an additional acidic residue in the multidrug recognition pocket of MdfA. This outcome further illustrates the exceptional promiscuous capabilities of multidrug transporters.
Project description:Multidrug resistance is a serious threat to public health. Proton motive force-driven antiporters from the major facilitator superfamily (MFS) constitute a major group of multidrug-resistance transporters. Currently, no reports on crystal structures of MFS antiporters in complex with their substrates exist. The E. coli MdfA transporter is a well-studied model system for biochemical analyses of multidrug-resistance MFS antiporters. Here, we report three crystal structures of MdfA-ligand complexes at resolutions up to 2.0 Å, all in the inward-facing conformation. The substrate-binding site sits proximal to the conserved acidic residue, D34. Our mutagenesis studies support the structural observations of the substrate-binding mode and the notion that D34 responds to substrate binding by adjusting its protonation status. Taken together, our data unveil the substrate-binding mode of MFS antiporters and suggest a mechanism of transport via this group of transporters.
Project description:The active efflux of antibiotics by multidrug-resistance (MDR) transporters is a major pathway of drug resistance and complicates the clinical treatment of bacterial infections. MdfA is a member of the major facilitator superfamily (MFS) from Escherichia coli and provides resistance to a wide variety of dissimilar toxic compounds, including neutral, cationic and zwitterionic substances. The 12-transmembrane-helix MdfA was expressed as a GFP-octahistidine fusion protein with a TEV protease cleavage site. Following tag removal, MdfA was purified using two chromatographic steps, complexed with a Fab fragment and further purified using size-exclusion chromatography. MdfA and MdfA-Fab complexes were subjected to both vapour-diffusion and lipidic cubic phase (LCP) crystallization techniques. Vapour-diffusion-grown crystals were of type II, with poor diffraction behaviour and weak crystal contacts. LCP lipid screening resulted in type I crystals that diffracted to 3.4?Å resolution and belonged to the hexagonal space group P6122.
Project description:MdfA is a prototypical H+-coupled multidrug transporter that is characterized by extraordinarily broad substrate specificity. The involvement of specific H-bonds in MdfA-drug interactions and the simplicity of altering the substrate specificity of MdfA contradict the promiscuous nature of multidrug recognition, presenting a baffling conundrum. Here we show the X-ray structures of MdfA variant I239T/G354E in complexes with three electrically different ligands, determined at resolutions up to 2.2?Å. Our structures reveal that I239T/G354E interacts with these compounds differently from MdfA and that I239T/G354E possesses two discrete, non-overlapping substrate-binding sites. Our results shed new light on the molecular design of multidrug-binding and protonation sites and highlight the importance of often-neglected, long-range charge-charge interactions in multidrug recognition. Beyond helping to solve the ostensible conundrum of multidrug recognition, our findings suggest the mechanistic difference between substrate and inhibitor for any H+-dependent multidrug transporter, which may open new vistas on curtailing efflux-mediated multidrug resistance.
Project description:Methodological and technological advances in EPR spectroscopy have enabled novel insight into the structural and dynamic aspects of integral membrane proteins. In addition to an extensive toolkit of EPR methods, multiple spin labels have been developed and utilized, among them Gd(III)-chelates which offer high sensitivity at high magnetic fields. Here, we applied a dual labeling approach, employing nitroxide and Gd(III) spin labels, in conjunction with Q-band and W-band double electron-electron resonance (DEER) measurements to characterize the solution structure of the detergent-solubilized multidrug transporter MdfA from E. coli. Our results identify highly flexible regions of MdfA, which may play an important role in its functional dynamics. Comparison of distance distribution of spin label pairs on the periplasm with those calculated using inward- and outward-facing crystal structures of MdfA, show that in detergent micelles, the protein adopts a predominantly outward-facing conformation, although more closed than the crystal structure. The cytoplasmic pairs suggest a small preference to the outward-facing crystal structure, with a somewhat more open conformation than the crystal structure. Parallel DEER measurements with the two types of labels led to similar distance distributions, demonstrating the feasibility of using W-band spectroscopy with a Gd(III) label for investigation of the structural dynamics of membrane proteins.
Project description:The largest family of solute transporters includes ion motive force-driven secondary transporters. Several well characterized solute-specific transport systems in this group have at least one irreplaceable acidic residue that plays a critical role in energy coupling during transport. Previous studies have established the importance of acidic residues in substrate recognition by major facilitator superfamily secondary multidrug transporters, but their role in the transport mechanism remained unknown. We have been investigating the involvement of acidic residues in the mechanism of MdfA, an Escherichia coli secondary multidrug/proton antiporter. We demonstrated that no single negatively charged side chain plays an irreplaceable role in MdfA. Accordingly, we hypothesized that MdfA might be able to utilize at least two acidic residues alternatively. In this study, we present evidence that indeed, unlike solute-specific secondary transporters, MdfA tolerates displacements of an essential negative charge to various locations in the putative drug translocation pathway. The results suggest that MdfA utilizes a proton translocation strategy that is less sensitive to perturbations in the geometry of the proton-binding site, further illustrating the exceptional structural promiscuity of multidrug transporters.
Project description:In a single quantitative study, we measured acrA, acrB, tolC, mdfA, and norE expression in Escherichia coli clinical isolates by using real-time PCR. acrA and acrB overexpression strongly correlated with fluoroquinolone and multidrug resistance; tolC, mdfA, and norE expression did not. The order of abundance of efflux pump transcripts in all fluoroquinolone-susceptible isolates was tolC (highest), then acrA and acrB, and then mdfA and norE. Our findings suggest acrAB overexpression is an indicator of multidrug resistance.
Project description:The DHA12 family of transporters contains a number of prokaryotic and eukaryote membrane proteins. Some of these proteins share conserved sites intrinsic to substrate recognition, structural stabilization and conformational changes. For this study, we chose the MdfA transporter as a model DHA12 protein to study some general characteristics of the vesicular neurotransmitter transporters (VNTs), which all belong to the DHA12 family. Two crystal structures were produced for E. coli MdfA, one in complex with acetylcholine and the other with potential reserpine, which are substrate and inhibitor of VNTs, respectively. These structures show that the binding sites of these two molecules are different. The Ach-binding MfdA is mainly dependent on D34, while reserpine-binding site is more hydrophobic. Based on sequence alignment and homology modelling, we were able to provide mechanistic insights into the association between the inhibition and the conformational changes of these transporters.
Project description:Bacterial resistance to antibiotics and biocides is an increasing public health problem. Genes encoding integral membrane proteins belonging to the DedA family are present in most bacterial genomes, including Escherichia coli. An E. coli strain lacking partially redundant DedA family genes yqjA and yghB (strain BC202) displays temperature sensitivity and cell division defects. These phenotypes can be corrected by overexpression of mdfA, an Na(+)-K(+)/H(+) antiporter of the major facilitator superfamily. We show that BC202 is hypersensitive to several biocides and cationic compounds that are known substrates of several multidrug resistance transporters, including MdfA, EmrE, and AcrB. The introduction of deletions of genes encoding these drug transporters into BC202 results in additional sensitivity. Expression of wild-type yghB or yqjA can restore drug resistance, but this is eliminated upon mutation of two membrane-embedded acidic amino acids (E39 or D51 in either protein). This dependence upon membrane-embedded acidic amino acids is a hallmark of proton-dependent antiporters. Overexpression of mdfA in BC202 or artificially restoring proton motive force (PMF) restores wild-type resistance to substrates of MdfA as well as other drug resistance transporters such as EmrE and AcrAB. These results suggest that YqjA and YghB may be membrane transporters required for PMF-dependent drug efflux in E. coli.
Project description:Multidrug resistance (MDR) poses a major challenge to medicine. A principle cause of MDR is through active efflux by MDR transporters situated in the bacterial membrane. Here we present the crystal structure of the major facilitator superfamily (MFS) drug/H+ antiporter MdfA from Escherichia coli in an outward open conformation. Comparison with the inward facing (drug binding) state shows that, in addition to the expected change in relative orientations of the N- and C-terminal lobes of the antiporter, the conformation of TM5 is kinked and twisted. In vitro reconstitution experiments demonstrate the importance of selected residues for transport and molecular dynamics simulations are used to gain insights into antiporter switching. With the availability of structures of alternative conformational states, we anticipate that MdfA will serve as a model system for understanding drug efflux in MFS MDR antiporters.