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Dissection of Mechanistic Principles of a Secondary Multidrug Efflux Protein
Abstract
Multidrug transporters are ubiquitous efflux pumps that provide cells with defense against various toxic compounds. In bacteria, which typically harbor numerous multidrug transporter genes, the majority function as secondary multidrug/proton antiporters. Proton-coupled secondary transport is a fundamental process that is not fully understood, largely owing to the obscure nature of proton-transporter interactions. Here we analyzed the substrate/proton coupling mechanism in MdfA, a model multidrug/proton antiporter. By measuring the effect of protons on substrate binding and by directly measuring proton binding and release, we show that substrates and protons compete for binding to MdfA. Our studies strongly suggest that competition is an integral feature of secondary multidrug transport. We identified the proton-binding acidic residue and show that, surprisingly, the substrate binds at a different site. Together, the results suggest an interesting mode of indirect competition as a mechanism of multidrug/proton antiport.
... The feasibility of such a mechanism is dependent on the competition between substrate and protons for binding to the transporter (Schuldiner, 2014), which in turn is mediated by tuning of the pK a of the carboxyl groups of one or more highly conserved acidic residues located within or near the substrate binding site in the membrane-embedded regions of the protein (Adler et al., 2004;Sigal et al., 2006). A previous study of the model Escherichia coli MFS multidrug efflux antiporter MdfA demonstrated that substrates and protons compete for binding to the protein (Fluman et al., 2012). The present study investigated the effects of proton concentration on binding of antimicrobial substrate tetraphenylphosphonium (TPP þ ) to the E. coli MFS multidrug/H þ antiporter MdtM and provides additional supporting evidence that the competition for binding between drug substrate and protons is a general feature of secondary multidrug efflux. ...
... Competition between protons and substrate is regarded as integral to the catalytic transport activity of the electrochemical proton gradient-driven MFS antiporters. In the MFS drug/H þ antiporter MdfA, the whole process is modulated by protonation of two conserved, membrane-embedded acidic residues; a glutamate at position 26 and aspartate at position 34 (Fluman et al., 2012). MdtM possesses two membrane-embedded aspartate residues at positions 22 and 30 (Holdsworth & Law, 2012) and it is pertinent to speculate that these residues represent the protonation sites in that transporter. ...
... The nature of the competition between substrate and protons can vary between MFS family members. In MdfA, this competition is allosteric with protons and TPP þ binding to different sites in the protein (Fluman et al., 2012). It is likely that the same mutually exclusive binding of protons and substrate is a feature of MdtM. ...
Proton electrochemical gradient-driven multidrug efflux activity of representatives of the major facilitator superfamily (MFS) of secondary active transporters contributes to antimicrobial resistance of pathogenic bacteria. Integral to the mechanism of these transporters is a proposed competition between substrate and protons for the binding site of the protein. The current work investigated the competition between protons and antimicrobial substrate for binding to the Escherichia coli MFS multidrug/H ⁺ antiporter MdtM by measuring the quench of intrinsic protein fluorescence upon titration of substrate tetraphenylphosphonium into a solution of purified MdtM over a range of pH values between pH 8.8 and 5.9. The results, which revealed that protons inhibit binding of substrate to MdtM in a competitive manner, are consistent with those reported in a study on the related MFS multidrug/H ⁺ antiporter MdfA and provide further evidence that competition for binding between substrate and protons is a general feature of secondary multidrug efflux.
... In antiporters, however, substrate must be released prior to binding and translocation of the counter-transported ion. 361 By preventing binding of the counter substrates together, the "Ping-Pong"-like mechanism of antiporters prevents cotransport of both substrates in the same direction. 361 In RND transporters, the separation of the proton and the substrate-binding sites further enforces an antiport mechanism. ...
... 361 By preventing binding of the counter substrates together, the "Ping-Pong"-like mechanism of antiporters prevents cotransport of both substrates in the same direction. 361 In RND transporters, the separation of the proton and the substrate-binding sites further enforces an antiport mechanism. A TM carboxylic residue is thought to be protonated during the transition of the transporter from the Binding to Extrusion state, and the proton transfer is needed to reset the transporter from the Extrusion to Access conformation. ...
... 286 Different transporters work with distinct proton:substrate stoichiometries. Although a stoichiometry of one proton per substrate was observed with LacY and MdfA from E. coli, 361 QacA from S. aureus, and LmrP from L. lactis use stoichiometries of up to 2 and 3, respectively. 362,363 Furthermore, in some transporters, e.g., GlpT that functions to couple an outward flow of internal inorganic phosphate (Pi) to the uptake of glycerol-3-phosphate (G3P) ( Table 4), the substrate stoichiometry is also pH-dependent, with a 2:1 Pi:sugar phosphate exchange measured at pH 7 and a 1:1 exchange measured at pH 5.2. ...
Cell envelope plays a dual role in the life of bacteria by simultaneously protecting it from a hostile environment and facilitating access to beneficial molecules. At the heart of this ability lie the restrictive properties of the cellular membrane augmented by efflux transporters, which preclude intracellular penetration of most molecules except with the help of specialized uptake mediators. Recently, kinetic properties of the cell envelope came into focus driven on one hand by the urgent need in new antibiotics and, on the other hand, by experimental and theoretical advances in studies of transmembrane transport. A notable result from these studies is the development of a kinetic formalism that integrates the Michaelis–Menten behavior of individual transporters with transmembrane diffusion and offers a quantitative basis for the analysis of intracellular penetration of bioactive compounds. This review surveys key experimental and computational approaches to the investigation of transport by individual translocators and in whole cells, summarizes key findings from these studies and outlines implications for antibiotic discovery. Special emphasis is placed on Gram-negative bacteria, whose envelope contains two separate membranes. This feature sets these organisms apart from Gram-positive bacteria and eukaryotic cells by providing them with full benefits of the synergy between slow transmembrane diffusion and active efflux.
... To understand the molecular mechanism of proton coupling and antiport, several biochemical and structural investigations have been conducted [19e22]. In antiporters, substrate binding and protonation are often found to compete with each other [23]. Substrate binding-induced deprotonation is thought to trigger the conformational change from inward-facing to outwardfacing. ...
... Among them, the structure of MdfA, a well-studied prototype multidrug-resistance transporter [26], has been solved in two different conformations, including substratebound inward-facing and outward-open [20,25]. Biochemical studies of MdfA confirmed competition between substrate and protons in secondary multidrug transport [23]. However, the structural basis of the alternating access mechanism of DHA transport remains to be elucidated. ...
... Substrate binding and protonation are found to compete with each other in MdfA [23]. This suggests that there may be competition between substrate binding and protonation of residues around substrate-binding sites in DHA antiporters and the deprotonation of titratable residues in the cavity is an essential step for substrate binding. ...
Drug-proton antiporters (DHA) play an important role in multi-drug resistance, utilizing the proton-motive force to drive the expulsion of toxic molecules, including antibiotics and drugs. DHA transporters belong to the major facilitator superfamily (MFS), members of which deliver substrates by utilizing the alternating access model of transport. However, the transport process is still elusive. Here, we report the structures of SotB, a member of DHA1 family (TCDB: 2.A.1.2) from Escherichia coli. Four crystal structures of SotB were captured in different conformations, including substrate-bound occluded, inward-facing, and inward-open states. Comparisons between the four structures reveal nonlinear rigid-body movements of alternating access during the state transition from inward-open to occluded conformation. This work not only reveals the conformational dynamics of SotB but also deepens our understanding of the alternating access mechanism of MFS transporters.
... 29 ), which is similar to the experimentally measured pK a of 6.5 (ref. 19 ). Thus, given the pH (8.0) of the crystal solutions, our Cm-bound structure of E26T/D34M/A150E likely portrays a substrate-bound, deprotonated transporter. ...
... Since E26T/D34M/A150E is an H + -coupled multidrug transporter 19 , we next asked if it interacts with Cm differently when the experimental pH is lowered below the pK a of A150E. Hence, we determined the Cm-bound structure of E26T/D34M/A150E at pH 5.0 (Supplementary Table 1), which likely represents a substrate-bound, protonated transporter. ...
... Thirdly, the observation of a Cm-bound, protonated E26T/D34M/A150E, i.e., a fully loaded intermediate state (Fig. 1c), is unexpected, since it was established that the binding of substrate and H + to MdfA is mutually exclusive 19,26 . However, the Cm-bound structure of protonated E26T/D34M/A150E is functionally relevant because the mutations of the substrate-binding protein residues exerted deleterious effects on the transport function (Fig. 2). ...
The rapid increase of multidrug resistance poses urgent threats to human health. Multidrug transporters prompt multidrug resistance by exporting different therapeutics across cell membranes, often by utilizing the H+ electrochemical gradient. MdfA from Escherichia coli is a prototypical H+ -dependent multidrug transporter belonging to the Major Facilitator Superfamily. Prior studies revealed unusual flexibility in the coupling between multidrug binding and deprotonation in MdfA, but the mechanistic basis for this flexibility was obscure. Here we report the X-ray structures of a MdfA mutant E26T/D34M/A150E, wherein the multidrug-binding and protonation sites were revamped, separately bound to three different substrates at resolutions up to 2.0 Å. To validate the functional relevance of these structures, we conducted mutational and biochemical studies. Our data elucidated intermediate states during antibiotic recognition and suggested structural changes that accompany the substrate-evoked deprotonation of E26T/D34M/A150E. These findings help to explain the mechanistic flexibility in drug/H+ coupling observed in MdfA and may inspire therapeutic development to preempt efflux-mediated antimicrobial resistance.
... The DedA family includes E. coli YqjA and YghB; putative proton dependent transporters that together are required for normal growth and cell division (Thompkins et al., 2008;Sikdar and Doerrler, 2010) and resistance to a number of antibiotics and biocides (Kumar and Doerrler, 2014) while YqjA is alone required for alkaline tolerance (Kumar and Doerrler, 2015). Both YqjA and YghB possess essential membrane embedded charged amino acids (Kumar and Doerrler, 2014;Kumar et al., 2016) that are present in proton-dependent transporters belonging to the major facilitator superfamily and other families (Noumi et al., 1997;Abramson et al., 2004;Adler and Bibi, 2004;Sigal et al., 2005;Fluman et al., 2012;Holdsworth and Law, 2012). While the reasons for this are unclear, DedA family proteins are required for polymyxin and/or antimicrobial peptide resistance of Salmonella enterica (Shi et al., 2004), Neisseria meningitidis (Tzeng et al., 2005), E. coli (Weatherspoon-Griffin et al., 2011), K. pneumoniae (Jana et al., 2017) and Enterobacter cloacae . ...
... This conservation includes charged amino acids E67, D79, R161, and R167 that are in comparable positions as YghB/YqjA E39, D51, R130 and R136, respectively (Figure 2), which are required for function (Kumar and Doerrler, 2014;Kumar et al., 2016). Acidic amino acids have been shown to be required for function of numerous secondary transporters including MdfA, NhaA, MdtM, and LacY (Gerchman et al., 1993;Noumi et al., 1997;Abramson et al., 2004;Adler and Bibi, 2004;Sigal et al., 2005;Fluman et al., 2012;Holdsworth and Law, 2012). Furthermore, the significance of membrane embedded basic amino acids such as arginine are well documented in the literature and play a role in a number of processes including regulation of redox potential FIGURE 2 | Amino acid similarity between Escherichia coli and B. thailandensis DedA family proteins. ...
... Undecaprenyl-P produced is recycled to the cytoplasm possibly by UppP/BacA, the undecaprenyl pyrophosphate phosphatase, which bears similarity to MdfA and other transporters (El Ghachi et al., 2018;Workman et al., 2018). Both MdfA (Fluman et al., 2012) and EmrE (Robinson et al., 2017) are proton-dependent transporters suggesting that the PMF may be required for both the transbilayer movement of undecaprenyl-P-Ara4N and the recycling of undecaprenyl-P. Perturbation of the PMF in DedA family mutants may interfere with either or both of these steps. ...
Colistin is a “last resort” antibiotic for treatment of infections caused by some multidrug resistant Gram-negative bacterial pathogens. Resistance to colistin varies between bacterial species. Some Gram-negative bacteria such as Burkholderia spp. are intrinsically resistant to very high levels of colistin with minimal inhibitory concentrations often above 0.5 mg/ml. We have previously shown DedA family proteins YqjA and YghB are conserved membrane transporters required for alkaline tolerance and resistance to several classes of dyes and antibiotics in Escherichia coli. Here, we show that a DedA family protein in B. thailandensis (DbcA; DedA of Burkholderia required for colistin resistance) is a membrane transporter required for resistance to colistin. Mutation of dbcA results in >100-fold greater sensitivity to colistin. Colistin resistance is often conferred via covalent modification of lipopolysaccharide (LPS) lipid A. Mass spectrometry of lipid A of ∆dbcA showed a sharp reduction of aminoarabinose in lipid A compared to wild type. Complementation of colistin sensitivity of B. thailandensis ∆dbcA was observed by expression of dbcA, E. coli yghB or E. coli yqjA. Many proton-dependent transporters possess charged amino acids in transmembrane domains that take part in the transport mechanism and are essential for function. Site directed mutagenesis of conserved and predicted membrane embedded charged amino acids suggest that DbcA functions as a proton-dependent transporter. Direct measurement of membrane potential shows that B. thailandensis ΔdbcA is partially depolarized suggesting that loss of protonmotive force can lead to alterations in LPS structure and severe colistin sensitivity in this species.
... MdfA exhibits an extremely broad spectrum of drug recognition and can couple the export of cationic, neutral, and zwitterionic compounds to the import of H + , with a drug/H + stoichiometry of 1:1 (refs. [9][10][11] ). The membrane-embedded D34 in MdfA appears to serve as the protonation site 11 . ...
... [9][10][11] ). The membrane-embedded D34 in MdfA appears to serve as the protonation site 11 . Moreover, previous studies identified a number of MdfA mutants with intriguingly different transport properties from those of the wild type protein [12][13][14][15][16][17][18] , including I239T/ G354E. ...
... We then mutated some of the LDAO-interacting amino acids identified from the Q131R structure ( Supplementary Fig. 4), and tested the function of these single mutants in the LDAO susceptibility assay. Bacteria expressing both the vector and E26T/D34M, an inactive MdfA mutant 11 , were used to measure the background level of cellular resistance to LDAO, which suggested that the endogenous efflux transporters exerted negligible effect on the resistance assay ( Supplementary Fig. 3). Moreover, we found that the mutations of Y30, N33, D34, M58, and L236 abrogated the ability of MdfA to confer LDAO resistance to E. coli. ...
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.
... Like SotB, MdfA derived from E. coli also belongs to the MFS Mdr transporter, which can transport a variety of substances (Edgar and Bibi, 1997;Fluman et al., 2012). The sequence similarity between SotB and MdfA is 13.4% (Supplementary Table S2). ...
... The SotB2 structure (rank_1_model_2) produced by AlphaFold 2 is in the inwardoccluded conformation, which contains the typical MFS fold of two six-helix bundles (N and C domains) with a central cavity, each 6-TMs bundle is itself made up of two 3-TMs segments related by a 180°rotation running parallel to the plane of the membrane (Figures 7B,C). MFS Mdr transporter MdfA exhibits an extremely broad spectrum of drug recognition (Edgar and Bibi, 1997;Fluman et al., 2012). The superimposition of the whole structure of SotB, SotB2, and MdfA revealed that the three structures are very similar overall ( Figure 7E), the r.m.s.d. between SotB and SotB2, SotB and MdfA, SotB2 and MdfA is 3.3 Å over 363 residues, 2.8 Å over 359 residues and 3.6 Å over 345 residues, respectively, and the most significant differences were observed in the flexible loops and the SotB2 has a longer TM12 in the C-terminal domain compared with SotB and MdfA ( Figure 7E). ...
Dysfunction of the major facilitator superfamily multidrug (MFS Mdr) transporters can lead to a variety of serious diseases in human. In bacteria, such membrane proteins are often associated with bacterial resistance. However, as one of the MFS Mdr transporters, the physiological function of SotB from Escherichia coli is poorly understood to date. To better understand the function and mechanism of SotB, a systematic study on this MFS Mdr transporter was carried out. In this study, SotB was found to directly efflux L-arabinose in E. coli by overexpressing sotB gene combined with cell based radiotracer uptake assay. Besides, the surface plasmon resonance (SPR) studies, the L-arabinose inhibition assays, together with precise molecular docking analysis, reveal the following: (i) the functional importance of E29 (protonation), H115/N343 (substrate recognition), and W119/S339 (substrate efflux) in the SotB mediated export of L-arabinose, and (ii) for the first time find that D-xylose, an isomer of L-arabinose, likely hinders the binding of L-arabinose with SotB as a competitive inhibitor. Finally, by analyzing the structure of SotB2 (shares 62.8% sequence similarity with SotB) predicted by AlphaFold 2, the different molecular mechanism of substrate recognition between SotB and SotB2 is explained. To our knowledge, this is the first systematic study of MFS Mdr transporter SotB. The structural information, together with the biochemical inspections in this study, provide a valuable framework for further deciphering the functional mechanisms of the physiologically important L-arabinose transporter SotB and its family.
... It transports lipophilic, cationic, and neutral substrates, in each case driven by the proton motive force 9,10 . Two acidic residues within TM1, Glu26 TM1 and Asp34 TM1 , have been implicated in proton (H + ) and substrate transport coupling [11][12][13] , and it has been proposed that changes in their protonation could lead to local structural changes within the binding pocket upon H + /substrate binding 11 . The recently reported structure of chloramphenicol-bound MdfA in an inward facing (I f ) conformation 14 reveals the antibiotic bound in the immediate vicinity of Asp34 TM1 , in line with earlier biochemical data 12,13 . ...
... Two acidic residues within TM1, Glu26 TM1 and Asp34 TM1 , have been implicated in proton (H + ) and substrate transport coupling [11][12][13] , and it has been proposed that changes in their protonation could lead to local structural changes within the binding pocket upon H + /substrate binding 11 . The recently reported structure of chloramphenicol-bound MdfA in an inward facing (I f ) conformation 14 reveals the antibiotic bound in the immediate vicinity of Asp34 TM1 , in line with earlier biochemical data 12,13 . ...
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.
... A motif (termed motif C) within the TMH5 situated in the interface between the two amino-terminal and carboxy-terminal bundles could participate with other helices to prevent proton leakage. Two conserved acidic residues, E26 and D34, have been identified in motif D of MdfA, and those residues were shown to be involved in proton transfer 45 , although only D34 participates in substrate binding 40 . Spectroscopic data suggest that protonation drives the conformational switch between alternating access states in the antiporters MdfA and LmrP 46 . ...
... MdfA also transports divalent cations with two charged moieties separated by a long linker; however, the export of such a cation drug molecule needs two consecutive transport cycles 50 . MdfA shows an indirect competition mechanism between H + and substrates 45 , whereas both direct and indirect mechanisms of competition might be relevant to the case of LmrP 51 . ...
Infections arising from multidrug-resistant pathogenic bacteria are spreading rapidly throughout the world and threaten to become untreatable. The origins of resistance are numerous and complex, but one underlying factor is the capacity of bacteria to rapidly export drugs through the intrinsic activity of efflux pumps. In this Review, we describe recent advances that have increased our understanding of the structures and molecular mechanisms of multidrug efflux pumps in bacteria. Clinical and laboratory data indicate that efflux pumps function not only in the drug extrusion process but also in virulence and the adaptive responses that contribute to antimicrobial resistance during infection. The emerging picture of the structure, function and regulation of efflux pumps suggests opportunities for countering their activities.
... 12,22,24 However, the termini of TMS 12 remained to be experimentally validated. Labelling of cysteine mutants with sulphhydrylreactive reagents such as FM has been used to experimentally probe whether a residue is located in the hydrophobic core of membrane bilayer (unreactive to labelling) or in a surface hydrophilic environment (able to be labelled) and therefore enabling the assignment of TMS in transporters, [25][26][27][28] including QacA. 15,18 The FM reactivity levels of the 38 cysteine-substituted QacA mutants in this study were compared with that of a QacA P309C mutant, previously shown to be highly reactive with FM and therefore located outside of the membrane environment. ...
Objectives
To elucidate the importance of a region in QacA predicted to be important in antimicrobial substrate recognition.
Methods
A total of 38 amino acid residues within or flanking putative transmembrane helix segment (TMS) 12 of QacA were individually replaced with cysteine using site-directed mutagenesis. The impact of these mutations on protein expression, drug resistance, transport activity and interaction with sulphhydryl-binding compounds was determined.
Results
Accessibility analysis of cysteine-substituted mutants identified the extents of TMS 12, which allowed for refinement of the QacA topology model. Mutation of Gly-361, Gly-379 and Ser-387 in QacA resulted in reduced resistance to at least one bivalent substrate. Interaction with sulphhydryl-binding compounds in efflux and binding assays demonstrated the role of Gly-361 and Ser-387 in the binding and transport pathway of specific substrates. The highly conserved residue Gly-379 was found to be important for the transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and interhelical interactions.
Conclusions
TMS 12 and its external flanking loop is required for the structural and functional integrity of QacA and contains amino acids directly involved in the interaction with substrates.
... According to the alternative access mechanism, SLC19A1 would cycle between the inward-facing and outward-facing conformations to carry its substrates across the cell membrane 41 . In our current structures, the extracellular (5-MTHF) and intracellular (TPP) substrates are bound to the identical site in SLC19A1, similar to that observed in some other antiporters 42,43 . It is plausible that cytosolic TPP could liberate 5-MTHF from the inward-facing SLC19A1 through competition under physiological conditions, and SLC19A would then adopt the outward-facing conformation for releasing TPP and binding extracellular 5-MTHF again (Fig. 5). ...
Folate (vitamin B9) is the coenzyme involved in one-carbon transfer biochemical reactions essential for cell survival and proliferation, with its inadequacy causing developmental defects or severe diseases. Notably, mammalian cells lack the ability to de novo synthesize folate but instead rely on its intake from extracellular sources via specific transporters or receptors, among which SLC19A1 is the ubiquitously expressed one in tissues. However, the mechanism of substrate recognition by SLC19A1 remains unclear. Here we report the cryo-EM structures of human SLC19A1 and its complex with 5-methyltetrahydrofolate at 3.5–3.6 Å resolution and elucidate the critical residues for substrate recognition. In particular, we reveal that two variant residues among SLC19 subfamily members designate the specificity for folate. Moreover, we identify intracellular thiamine pyrophosphate as the favorite coupled substrate for folate transport by SLC19A1. Together, this work establishes the molecular basis of substrate recognition by this central folate transporter.
... According to the alternative access mechanism, SLC19A1 would cycle between the inward-facing and outward-facing conformations to carry its substrates across the cell membrane 41 . In our current structures, the extracellular (5-MTHF) and intracellular (TPP) substrates are bound to the identical site in SLC19A1, similar to that observed in some other antiporters 42,43 . It is plausible that cytosolic TPP could liberate 5-MTHF from the inward-facing SLC19A1 through competition under physiological conditions, and SLC19A would then adopt the outward-facing conformation for releasing TPP and binding extracellular 5-MTHF again (Fig. 5). ...
Folate (vitamin B9) is the coenzyme involved in one-carbon transfer biochemical reactions essential for cell survival and proliferation, with its inadequacy causing developmental defects or severe diseases. Notably, mammalian cells lack the ability to de novo synthesize folate but instead rely on its intake from extracellular sources via specific transporters or receptors, among which SLC19A1 is the ubiquitously expressed one in tissues. However, the mechanism of substrate recognition by SLC19A1 has been unclear. Here we report the cryo-EM structures of human SLC19A1 and its complex with 5-methyltetrahydrofolate at 3.5-3.6 angstrom resolution and elucidate the critical residues for substrate recognition. In particular, we reveal that two variant residues among SLC19 subfamily members would designate the specificity for folate. Moreover, we identify intracellular thiamine pyrophosphate as the favorite coupled substrate for folate transport by SLC19A1. Together, this work has established the molecular basis of substrate recognition by this central folate transporter.
... Further studies suggest that although proton translocation and substrate transport occur in distinct sites, they always compete for protein binding. Consequently, protonation leads to conformation changes of the protein that facilitate substrate uptake from the intracellular side (inwardopen conformation), whereas deprotonation destabilizes the substrate-bound state of the protein and eventually leads to substrate release on the extracellular side (outward open conformation) [19,26,[48][49][50][51]. ...
The Major Facilitator Superfamily (MFS) drug:H + antiporter CaMdr1, from Candida albi-cans, is responsible for the efflux of structurally diverse antifungals. MFS members share a common fold of 12-14 transmembrane helices (TMHs) forming two N-and C-domains. Each domain is arranged in a pseudo-symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS proteins show great diversity in primary structure and few conserved signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously addressing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing CaMdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127ATMH1-> G140DTMH1) or 3-TMHs repeat (F216ATMH4-> G260ATMH5), at the hinge of 3-TMHs repeats tandems (R184ATMH3-> D235HTMH4, L480ATMH10-> A435TTMH9), and finally between the N-and C-domains (G230ATMH4-> P528HTMH12). Remarkably, most of these mutants belong to the different signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS proteins. Results also point to the specific role of TMH11 in the interplay between the N-and C-domains in the inward-to outward-open conformational transition.
... These residues are conserved in NorA's closest homologs, Bmr and Blt from Bacillus subtilis, yet are absent at corresponding positions in other DHA12 subfamily efflux pumps of known structure (EmrD, LmrP, MdfA and YajR 27,33-35 ; Fig. 2d). These efflux pumps instead contain essential aspartate and glutamate residues for proton-coupled transport at different membrane-embedded locations 27,36 . This observation highlights the subtle differences in transport mechanisms even among homologous drug efflux pumps. ...
Membrane protein efflux pumps confer antibiotic resistance by extruding structurally distinct compounds and lowering their intracellular concentration. Yet, there are no clinically approved drugs to inhibit efflux pumps, which would potentiate the efficacy of existing antibiotics rendered ineffective by drug efflux. Here we identified synthetic antigen-binding fragments (Fabs) that inhibit the quinolone transporter NorA from methicillin-resistant Staphylococcus aureus (MRSA). Structures of two NorA–Fab complexes determined using cryo-electron microscopy reveal a Fab loop deeply inserted in the substrate-binding pocket of NorA. An arginine residue on this loop interacts with two neighboring aspartate and glutamate residues essential for NorA-mediated antibiotic resistance in MRSA. Peptide mimics of the Fab loop inhibit NorA with submicromolar potency and ablate MRSA growth in combination with the antibiotic norfloxacin. These findings establish a class of peptide inhibitors that block antibiotic efflux in MRSA by targeting indispensable residues in NorA without the need for membrane permeability.
... Further studies suggest that although proton translocation and substrate transport occur in distinct sites, they always compete for protein binding. Consequently, protonation leads to conformation changes of the protein that facilitate substrate uptake from intracellular side (inward-open conformation) whereas deprotonation destabilizes the substrate-bound state of the protein and eventually leads to substrate release on the extracellular side (outward open conformation) (Yin et al. 2006;Schaedler and Van Veen 2010;Fluman et al. 2012;Heng et al. 2015;Wu et al. 2020;Drew et al. 2021b). ...
The Major Facilitator Superfamily (MFS) includes multiple families of proteins operating as uniporters, symporters and antiporters for a wide spectrum of substrates. Among them, the multidrug resistance-1 drug:H ⁺ antiporter Ca Mdr1 from Candida albicans is responsible for the efflux of structurally-diverse antifungals. MFS share a common fold of 12-14 transmembrane helices (TMHs) forming two N- and C-domains. Each domain is arranged in a pseudo symmetric fold of two tandems of 3-TMHs that alternatively expose the drug-binding site towards the inside or the outside of the yeast to promote drug binding and release. MFS show a high primary structure diversity and few conserved Signature motifs, each thought to have a common function in the superfamily, although not yet clearly established. Here, we provide new information on these motifs by having screened a library of 64 drug transport-deficient mutants and their corresponding suppressors spontaneously rescuing the deficiency. We found that five strains recovered the drug-resistance capacity by expressing Ca Mdr1 with a secondary mutation. The pairs of debilitating/rescuing residues are distributed either in the same TMH (T127A TMH1 ->G140D TMH1 ) or 3-TMHs repeat (F216A TMH4 ->G260A TMH5 ), at the hinge of 3-TMHs repeats tandems (R184A TMH3 ->D235H TMH4 , L480A TMH10 ->A435T TMH9 ), and finally between the N- and C-domains (G230A TMH4 ->P528H TMH12 ). Remarkably, most of these mutants belongs to the different Signature motifs, highlighting a mechanistic role and interplay thought to be conserved among MFS. Results point also to the specific role of TMH11 in the interplay between the N- and C-domains in the inward- to outward-open conformational transition.
... Tet (40) [197] Escherichia coli EmrB, EmrD e MdfA, MdtM e QacA [178,189,199,200,201] ...
The microorganism resistance to antibiotics has become one of the most worrying issues for science due to the difficulties related to clinical treatment and the rapid spread of diseases. Efflux pumps are classified into six groups of carrier proteins that are part of the different types of mechanisms that contribute to resistance in microorganisms, allowing their survival. The present study aimed to carry out a bibliographic review on the superfamilies of carriers in order to understand their compositions, expressions, substrates, and role in intrinsic resistance. At first, a search for manuscripts was carried out in the databases Medline, Pubmed, ScienceDirect, and Scielo, using as descriptors: efflux pump, expression, pump inhibitors and efflux superfamily. For article selection, two criteria were taken into account: for inclusion, those published between 2000 and 2020, including textbooks, and for exclusion, duplicates and academic collections. In this research, 139,615 published articles were obtained, with 312 selected articles and 7 book chapters that best met the aim. From the comprehensive analysis, it was possible to consider that the chromosomes and genetic elements can contain genes encoding efflux pumps and are responsible for multidrug resistance. Even though this is a well-explored topic in the scientific community, understanding the behavior of antibiotics as substrates that increase the expression of pump-encoding genes has challenged medicine. This review study succinctly summarizes the most relevant features of these systems, as well as their contribution to multidrug resistance.
... YqjA is required for growth of E. coli above pH 8.5 (10), similar to what was reported for MdfA (11) and other membrane transporters (12)(13)(14). Both YqjA and YghB possess essential membrane-embedded charged amino acids (4,15) that are present in proton-dependent transporters belonging to the MFS and RND families of efflux pumps (16)(17)(18)(19)(20)(21), suggesting key transport functions of the DedA family. ...
... YqjA is required for growth of E. coli above pH 8.5 (10), similar to what was reported for MdfA (11) and other membrane transporters (12)(13)(14). Both YqjA and YghB possess essential membrane-embedded charged amino acids (4,15) that are present in proton-dependent transporters belonging to the MFS and RND families of efflux pumps (16)(17)(18)(19)(20)(21), suggesting key transport functions of the DedA family. ...
... MFS transporters can confer resistance to a variety of toxic compounds, including specialized metabolites, fungicidal substances, and antibiotics (Sorbo et al., 2000;Fluman et al., 2012;Zhang et al., 2020). They can be used as drug H+ antiporters in microorganisms to indirectly regulate internal pH and stress response mechanisms in fungi (Santos et al., 2014); thus they can confer a multi-drug resistance (MDR) phenotype to fungi (Omrane et al., 2015). ...
The major facilitator superfamily (MFS) is one of the largest known membrane transporter families. MFSs are involved in many essential functions, but studies on the MFS family in poplar have not yet been reported. Here, we identified 41 MFS genes from Populus trichocarpa (PtrMFSs). We built a phylogenetic tree, which clearly divided members of PtrMFS into six groups with specific gene structures and protein motifs/domains. The promoter regions contain various cis-acting elements involved in stress and hormone responsiveness. Genes derived from segmental duplication events are unevenly distributed in 17 poplar chromosomes. Collinearity analysis showed that PtrMFS genes are conserved and homologous to corresponding genes from four other species. Transcriptome data indicated that 40 poplar MFS genes were differentially expressed when treated with Fusarium oxysporum. Co-expression networks and gene function annotations of MFS genes showed that MFS genes tightly co-regulated and closely related in function of transmembrane transport. Taken together, we systematically analyzed structure and function of genes and proteins in the PtrMFS family. Evidence indicated that poplar MFS genes play key roles in plant development and response to a biological stressor.
... A well-studied example of such behavior is MdfA that has two acidic residues E26 and D34 that mediate antibacterial efflux in this proton-coupled antiporter. While D34 is responsible for competitive protonsubstrate interactions, E26 located towards the cytosolic half of the vestibule can facilitate proton transfer via sequential binding of substrate and protons at D34 (51). The topology of LysO is distinct from MFS antiporters, nonetheless the proton-substrate coupling mechanisms could still retain similarities to MdfA within the transport vestibule of LysO. ...
LysO, a prototypical member of the LysO family, mediates export of L-lysine (Lys) and resistance to the toxic Lys antimetabolite, L-thialysine (Thl) in E. coli. Here, we have addressed unknown aspects of LysO function pertaining to its membrane topology and the mechanism by which it mediates Lys / Thl export. Using substituted cysteine (Cys) accessibility, here we delineated the membrane topology of LysO. Our studies support a model in which both the N- and C-termini of LysO are present at the periplasmic face of the membrane with a transmembrane (TM) domain comprising eight TM segments (TMSs) between them. In addition, a feature of intramembrane solvent exposure in LysO is inferred with the identification of membrane-located solvent-exposed Cys residues. Isosteric substitutions of a pair of conserved acidic residues, one E233, located in the solvent-exposed TMS7 and the other D261, in a solvent-exposed intramembrane segment located between TMS7 and TMS8, abolished LysO function in vivo. Thl, but not Lys, elicited proton release in inside-out membrane vesicles, a process requiring the presence of both E233 and D261. We postulate that Thl may be exported in antiport with H⁺, and that Lys may be a low-affinity export substrate. Our findings are compatible with a physiological scenario wherein in vivo LysO exports the naturally occurring antimetabolite Thl with higher affinity over the essential cellular metabolite Lys, thus affording protection from Thl toxicity and limiting wasteful export of Lys.
... YqjA is required for growth of E. coli above pH 8.5 (10), similar to what was reported for MdfA (11) and other membrane transporters (12)(13)(14). Both YqjA and YghB possess essential membrane-embedded charged amino acids (4,15) that are present in proton-dependent transporters belonging to the MFS and RND families of efflux pumps (16)(17)(18)(19)(20)(21), suggesting key transport functions of the DedA family. ...
Rice is an important source of food for more than half the world’s population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae . B. glumae synthesizes toxoflavin, an essential virulence factor, that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis , named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae Δ dbcA strain. In addition to polymyxin sensitivity, B. glumae Δ dbcA is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild type levels). Changes in membrane potential in B. glumae Δ dbcA were reproduced in the wild type strain by the addition of sub-inhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice suggesting a possible non-toxic chemical intervention for bacterial panicle blight.
IMPORTANCE
Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae . The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.
... TPP + interacts despite mutating the protonatable lysine in the vestibule (K398) suggesting that the compound, given its multiple phenyl groups, could interact at an allosteric site stabilizing the outward-open conformation of NorC. This is very different from competitive binding displayed by TPP + in antibacterial efflux transporters, MdfA and QacA, wherein substituting the primary protonation sites leads to a clear loss of TPP + interaction and transport propensity 34,37 . In the presence of the ICab, entry into the vestibule of NorC is clearly blocked causing a complete loss of interactions of the antibacterial compound TPP + with NorC ( Fig. 4a, b). ...
Transporters play vital roles in acquiring antimicrobial resistance among pathogenic bacteria. In this study, we report the X-ray structure of NorC, a 14-transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related transporters through the use of single-domain camelid antibodies.
... Two conserved acidic residues, E26 and D34, have been shown in MdfA to be involved in proton binding and release, and the negative charge on D34 is not essential for substrate binding as it could be replaced with asparagine without having an effect on in vitro substrate binding. 392 MdfA is broadly flexible in recognizing electroneutral or monovalent cationic drugs and catalyzes the efflux of these drugs in exchange with a single proton, and there appears to be an indirect competition mechanism between protons and substrates. However, MdfA was shown to also transport divalent cations where the charges were separated by a long linker. ...
Tripartite efflux pumps and the related type 1 secretion systems (T1SSs) in Gram-negative organisms are diverse in function, energization, and structural organization. They form continuous conduits spanning both the inner and the outer membrane and are composed of three principal components-the energized inner membrane transporters (belonging to ABC, RND, and MFS families), the outer membrane factor channel-like proteins, and linking the two, the periplasmic adaptor proteins (PAPs), also known as the membrane fusion proteins (MFPs). In this review we summarize the recent advances in understanding of structural biology, function, and regulation of these systems, highlighting the previously undescribed role of PAPs in providing a common architectural scaffold across diverse families of transporters. Despite being built from a limited number of basic structural domains, these complexes present a staggering variety of architectures. While key insights have been derived from the RND transporter systems, a closer inspection of the operation and structural organization of different tripartite systems reveals unexpected analogies between them, including those formed around MFS- and ATP-driven transporters, suggesting that they operate around basic common principles. Based on that we are proposing a new integrated model of PAP-mediated communication within the conformational cycling of tripartite systems, which could be expanded to other types of assemblies.
... In MdfA, the highly conserved TM1 acidic residues Glu26 and Asp34 are both required to achieve H + -coupled efflux of positively charged substrates, whereas only one out of these two residues are required to expel noncharged substrates. 217,220,314 While the substrate binding residues vary, all substrates directly form electrostatic interactions with Asp34 in TM1, which also serves as the principal H + -coupling residue ( Figure 16). Despite the variation of substrate-binding modes, mutation of substrate coordinating residues significantly reduces the efflux of monocationic compounds and neutrally charged drugs. ...
The major facilitator superfamily (MFS) is the largest known superfamily of secondary active transporters. MFS transporters are responsible for transporting a broad spectrum of substrates, either down their concentration gradient or uphill using the energy stored in the electrochemical gradients. Over the last 10 years, more than a hundred different MFS transporter structures covering close to 40 members have provided an atomic framework for piecing together the molecular basis of their transport cycles. Here, we summarize the remarkable promiscuity of MFS members in terms of substrate recognition and proton coupling as well as the intricate gating mechanisms undergone in achieving substrate translocation. We outline studies that show how residues far from the substrate binding site can be just as important for fine-tuning substrate recognition and specificity as those residues directly coordinating the substrate, and how a number of MFS transporters have evolved to form unique complexes with chaperone and signaling functions. Through a deeper mechanistic description of glucose (GLUT) transporters and multidrug resistance (MDR) antiporters, we outline novel refinements to the rocker-switch alternating-access model, such as a latch mechanism for proton-coupled monosaccharide transport. We emphasize that a full understanding of transport requires an elucidation of MFS transporter dynamics, energy landscapes, and the determination of how rate transitions are modulated by lipids.
... 23 D34 has also been demonstrated to serve as a proton binding site (alongside E26) and is thus a key residue in coupling substrate export to proton import. 24 Subsequent structural studies of MdfA have captured the transporter in an outwardfacing conformation (stabilized by forming a complex with an Fab antibody fragment), suggesting that MdfA mediates substrate transport using a classic MFS rocker-switch mechanism with an additional twisting of TM5. 25 In this transport model (Figure 1(b)), MdfA begins in an inward-facing conformation with D34 protonated. Substrate loading induces D34 deprotonation, freeing this residue to directly interact with the substrate via hydrogen bonding. ...
Infectious diseases present a major threat to public health globally. Pathogens can acquire resistance to anti-infectious agents via several means including transporter-mediated efflux. Typically, multidrug transporters feature spacious, dynamic, and chemically malleable binding sites to aid in the recognition and transport of chemically diverse substrates across cell membranes. Here, we discuss recent structural investigations of multidrug transporters involved in resistance to infectious diseases that belong to the ATP-binding cassette (ABC) superfamily, the major facilitator superfamily (MFS), the drug/metabolite transporter (DMT) superfamily, the multidrug and toxic compound extrusion (MATE) family, the small multidrug resistance (SMR) family, and the resistance-nodulation-division (RND) superfamily. These structural insights provide invaluable information for understanding and combatting multidrug resistance.
... Since Major facilitator superfamily (MFS) transporters are described for supplying resistance to various toxic compounds, including not only secondary metabolites but also fungicides and antibiotics (Del Sorbo et al., 2000;Fluman et al., 2012;Zhang et al., 2020), fungicide sensitivity assessment was carried out. The evaluation of the four genes through gene elimination and gene overexpression, highlighted that only PdMFS2 and PdMFS3 were able to contribute to fungicide resistance. ...
... Previously, we proposed that lower membrane potential observed in dbcA might affect the transport activity of undecaprenyl pyrophosphate [C 55 P(P)] phosphatase UppP/BacA (Panta et al., 2019), which bears similarity to MdfA, a prototypical H +coupled multidrug transporter, known to utilize PMF (Fluman et al., 2012). UppA/BacA has been shown to be involved in bacitracin resistance and is the C 55 P(P) phosphatase in E. coli (El Ghachi et al., 2004). ...
The DedA family is a conserved membrane protein family found in most organisms. A Burkholderia thailandensis DedA family protein, named DbcA, is required for high-level colistin (polymyxin E) resistance, but the mechanism awaits elucidation. Modification of lipopolysaccharide lipid A with the cationic sugar aminoarabinose (Ara4N) is required for colistin resistance and is dependent upon protonmotive force (PMF) dependent transporters. B. thailandensis dbcA lipid A contains only small amounts of Ara4N, likely leading to colistin sensitivity. Two B. thailandensis operons are required for lipid A modification with Ara4N, one needed for biosynthesis of undecaprenyl-P-Ara4N and one for transport of the lipid linked sugar and subsequent lipid A modification. Here, we directed overexpression of each arn operon by genomic insertion of inducible promoters. We found that overexpression of arn operons in dbcA can partially, but not completely, restore Ara4N modification of lipid A and colistin resistance. Artificially increasing the PMF by lowering the pH of the growth media also increased membrane potential, amounts of Ara4N, and colistin resistance of dbcA. In addition, the products of arn operons are essential for acid tolerance, suggesting a physiological function of Ara4N modification. Finally, we show that dbcA is sensitive to bacitracin and expression of a B. thailandensis UppP/BacA homolog (BTH_I1512) can partially restore resistance to bacitracin. Expression of a different UppP/BacA homolog (BTH_I2750) can partially restore colistin resistance, without changing the lipid A profile. This work suggests that maintaining optimal membrane potential at slightly alkaline pH media by DbcA is responsible for proper modification of lipid A by Ara4N and provides evidence of lipid A modification-dependent and-independent mechanisms of colistin resistance in B. thailandensis.
... Additionally, there are also acidic amino acids (E27 and E39) and basic amino acids (R117 and R123) within or in close to transmembrane spanning regions. These charged amino acids are important for the various proton-dependent secondary transporters, such as NhaA, MdtM (Fluman et al., 2012;Holdsworth and Law, 2012). What's more interesting, a recent report focusing on DedA family protein in Burkholderia thailandensis showed the membrane-embedded charged amino acids contributed to the colistin resistance (Panta et al., 2019). ...
Daptomycin (DAP) is a novel microbial lipopeptide antibiotic synthesized by the DAP biosynthetic gene cluster dpt of Streptomyces roseosporus (S. roseosporus). DptP gene locates upstream of dpt and confers DAP resistance to Streptomyces ambofaciens (S. ambofaciens). So far, the biological functions of dptP gene for S. roseosporus growth are still completely uncovered. We performed label‐free quantification proteomic dissections with loss‐ and gain‐of‐function experiments to decipher dptP‐involved functions. Deletion of dptP gene activated energy metabolism and metabolism of secondary metabolites pathways and enhanced the transcription levels and protein abundance of key members of the dpt cluster. Whereas dptP deletion inhibited transport/signal transduction and drug resistance pathways and protein abundance of cell division‐relative proteins, subsequently decreased mycelia cell growth rate. S. roseosporus strain with dptP deletion was more sensitive to DAP treatment compared to the wild type. In contrast, overexpression of dptP gene decreased transcription levels of DAP biosynthetic genes and enhanced growth rate of Streptomcyes strain upon elevated culture temperature and DAP supplementation. Taken together, dptP gene contributes to Streptomcyes primary growth under elevated temperature and DAP treatment, whereas it plays negative roles on metabolism of secondary metabolites and transcription of DAP biosynthetic genes.
... Furthermore, D27N mutant is known to be functionally inactive, as demonstrated in cell-based uptake assays 23 . However, such mutants have already been used successfully to decipher the molecular mechanism of other protoncoupled transporters such as the MDR transporters AcrB, LmrP, PfMATE and MdfA [33][34][35][36] , and identified key structural motifs during the transport cycle. Comparative HDX-MS experiments of protein harbouring protonation mimics appears to be a valuable method to study the molecular mechanism of proton-coupled transporters. ...
Proton-coupled transporters use transmembrane proton gradients to power active transport of nutrients inside the cell. High-resolution structures often fail to capture the coupling between proton and ligand binding, and conformational changes associated with transport. We combine HDX-MS with mutagenesis and MD simulations to dissect the molecular mechanism of the prototypical transporter XylE. We show that protonation of a conserved aspartate triggers conformational transition from outward-facing to inward-facing state. This transition only occurs in the presence of substrate xylose, while the inhibitor glucose locks the transporter in the outward-facing state. MD simulations corroborate the experiments by showing that only the combination of protonation and xylose binding, and not glucose, sets up the transporter for conformational switch. Overall, we demonstrate the unique ability of HDX-MS to distinguish between the conformational dynamics of inhibitor and substrate binding, and show that a specific allosteric coupling between substrate binding and protonation is a key step to initiate transport.
... TPP + interacts despite mutating the protonatable lysine in the vestibule (K398) suggesting that the compound, given its multiple phenyl groups, could interact at an allosteric site stabilizing the outward-open conformation of NorC. This is very different from competitive binding displayed by TPP + in antibacterial efflux transporters, MdfA and QacA, wherein substituting the primary protonation sites leads to a clear loss of TPP + interaction and transport propensity 34,37 . In the presence of the ICab, entry into the vestibule of NorC is clearly blocked causing a complete loss of interactions of the antibacterial compound TPP + with NorC ( Fig. 4a, b). ...
Multi-drug efflux is a major mechanism of acquiring antimicrobial resistance among superbugs. In this study, we report the X-ray structure of NorC, a 14 transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 Å. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related efflux pumps through the use of single- domain camelid antibodies.
... Since Major facilitator superfamily (MFS) transporters are described for supplying resistance to various toxic compounds, including not only secondary metabolites but also fungicides and antibiotics (Del Sorbo et al., 2000;Fluman et al., 2012;Zhang et al., 2020), fungicide sensitivity assessment was carried out. The evaluation of the four genes through gene elimination and gene overexpression, highlighted that only PdMFS2 and PdMFS3 were able to contribute to fungicide resistance. ...
Major facilitator superfamily (MFS) comprises a large family of fungal transporters. In this work four Penicillium digitatum MFS transporters named PdMFS2–5 were identified and functionally characterized through gene elimination and gene overexpression with aim of unveil the similarities and differences among members of the same family during pathogen-fruit interaction. Fungal mutants in which each of the MFS transporters were individually deleted, displayed a clear effect on their infective capacity during citrus fruit infection especially in two of them. In contrast, the observed effect on fungicide sensitivity limits PdMFS2 and PdMFS3 as transporters underlying fungicide resistance. Moreover, overexpression transformants confirmed P. digitatum MFS transporters function and PdMFS2 and PdMFS3 were able to confer fungicide resistance to P. digitatum strains originally fungicide sensitive. Gene transcription rate depended on each MFS transporter being PdMFS4 the one with higher gene expression. Transcriptional profiling was similar regardless the P. digitatum strain. The gene expression analysis showed an increase of PdMFSs transcription in all overexpression transformants, particularly in Pd27 strain. Expression analysis carried out during P. digitatum-citrus fruit interaction confirmed the contribution of all PdMFSs, excepting PdMFS5, in fungal virulence. These results indicate that MFS fungal transporters might be part of different processes and can replace other genes functions giving them a very high degree of versatility.
... (10) Results from biochemical studies suggested that a substrate must be released prior to binding of a proton, so there is a competition between binding a substrate and binding a proton. (11) Mechanistically, in the I f conformation residue Asp34 gets deprotonated, enabling the binding of a substrate in the multidrug recognition pocket. A conformational change towards the O o conformation is triggered by this binding. ...
MdfA, a member of the major facilitator superfamily (MFS), is a multidrug/proton antiporter from E. coli that has been considered a model for secondary multidrug (Mdr) transporters. Its transport mechanism, driven by a proton gradient, is associated with conformational changes, which accompany the recruitment of drugs and their release. In this work, we applied double-electron electron resonance (DEER) spectroscopy to locate the binding site of one of its substrates, tetraphenylphosphonium (TPP) within available crystal structures. We carried out Gd(III)-nitroxide distance measurements between MdfA labeled with a Gd(III) tag and the TPP analog mito-TEMPO (bearing the nitroxide moiety). Data were obtained both for MdfA solubilized in detergent micelles (n-dodecyl-β-D-maltopyranoside (DDM)), and reconstituted into lipid nanodiscs (ND). For both DDM and ND, the average position of the substrate at a neutral pH was found to be close to the ligand position in the I f (inward facing) crystal structure, with the DDM environment exhibiting a somewhat better agreement than the ND environment. We therefore conclude that the I f structure provides a good description for substrate-bound MdfA in DDM solution, while in ND the structure is slightly modified. A second binding site was found for the ND sample situated at the cytoplasmic side, towards the end of transmembrane helix 7 (TM7). In addition, we used DEER distance measurements on Gd(III) doubly labeled MdfA to track conformational changes within the periplasmic and cytoplasmic sides associated with substrate binding. We detected significant differences in the periplasmic side of MdfA, with the ND featuring a more closed conformation than in DDM, in agreement with earlier reports. The addition of TPP led to a noticeable conformational change in the periplasmic face in ND, attributed to a movement of TM10. This change was not observed in DDM.
Statement of Significance
MdfA is multidrug transporter from E. coli , which exhibits multidrug efflux activities with an unusually broad spectrum of drug specificities. While it has been established that solute transport by similar transporters is coupled to significant conformational changes, previous studies raised the possibility that this is not the case for MdfA. Moreover, it is not clear how MdfA functionally accommodates chemically dissimilar substrates. Towards resolving these open questions, we used double-electron electron resonance distance measurements to determine the binding site of a spin labeled drug analog within available crystal structures of MdfA and to examine how MdfA responds conformationally to drug binding. Moreover, we explored how these two are affected by the media, detergent micelles vs lipid nanodiscs.
... Charged amino acids play key roles in biological functions, including protein folding and binding interactions (Tollinger et al. 2003;Wells et al. 1987). In addition, acid/ base chemistry at charged residues are involved in enzyme catalysis such as protein degradation and proton transport across cellular membranes (Gayen et al. 2016;Morrison et al. 2015;Masureel 2014;Fluman et al. 2012;Fluman and Bibi 2009). Since the chemical shift is a sensitive reporter of the electrostatic environment, NMR spectroscopy has been a valuable experimental approach to probe site-specific protonation states and acid dissociation constants (pK a ) (Jehle 2006;Schmidt-Rohr et al. 2012;Reggie et al. 2011;Nielsen 2008;Oregioni et al. 2017;Pielak and Chou 1808;Hu 2006;Hu et al. 2012; Bartik et al. 1994;McIntosh 1996;Oda 1994). ...
NMR spectroscopy is commonly used to infer site-specific acid dissociation constants (pKa) since the chemical shift is sensitive to the protonation state. Methods that probe atoms nearest to the functional groups involved in acid/base chemistry are the most sensitive for determining the protonation state. In this work, we describe a magic-angle-spinning (MAS) solid-state NMR approach to measure chemical shifts on the side chain of the anionic residues aspartate and glutamate. This method involves a combination of double quantum spectroscopy in the indirect dimension and REDOR dephasing to provide a sensitive and resolved view of these amino acid residues that are commonly involved in enzyme catalysis and membrane protein transport. To demonstrate the applicability of the approach, we carried out measurements using a microcrystalline soluble protein (ubiquitin) and a membrane protein embedded in lipid bilayers (EmrE). Overall, the resolution available from the double quantum dimension and confidence in identification of aspartate and glutamate residues from the REDOR filter make this method the most convenient for characterizing protonation states and deriving pKa values using MAS solid-state NMR.
... 231 The substrates coupling in these transporters involve alternate binding and releasing of the substrate. 232 However, the conformational changes of the MFS proteins determine the transport mechanism. 233 The mutations in local repressor and global regulatory genes, mutations in the promoter of definite transporters, and insertional elements inserted upstream of transporters encoding genes cause overexpression of these efflux pumps. ...
Superbugs’ resistivity against available natural products has become an alarming global threat, causing a rapid deterioration in public health and claiming tens of thousands of lives yearly. Although the rapid discovery of small molecules from the plant and microbial origin with enhanced bioactivity has provided us with some hope, however, a rapid hike in the resistivity of superbugs has proven to be the biggest therapeutic hurdle of all times. Moreover, several distinct mechanisms endowed by these notorious superbugs make them immune to these antibiotics subsequently causing our antibiotic wardrobe to be obsolete. In this unfortunate situation, though the time-frame for discovering novel ‘hit molecules’ down the line remains largely unknown, our small hope and untiring efforts injected in hunting novel chemical scaffolds with unique molecular targets using high-throughput technologies may safeguard us against these life-threatening challenges to some extent. Amid this crisis, the current comprehensive review highlights the present status of knowledge, our search for bacteria Achilles’ heel, distinct molecular signaling that opportunistic pathogen bestows to trespass the toxicity of antibiotics, and facile strategies and appealing therapeutic targets of novel drugs. Herein, we also discuss multidimensional strategies to combat antimicrobial resistance.
... This fact underscores the need to develop new targets to achieve a more efficient control in citrus postharvest. In that sense, MFS transporters might be a good alternative since they could play a part in multidrug resistance in bacteria and fungi [29]. ...
A new Penicillium digitatum major facilitator superfamily (MFS) transporter (PdMFS1) was identified and functionally characterized in order to shed more light on the mechanisms underlying fungicide resistance. PdMFS1 can play an important role in the intensification of resistance to fungicides normally used in P. digitatum postharvest treatments. In the PdMFS1 disrupted mutants, a slight effect in response to chemical fungicides was observed, but fungicide sensitivity was highly affected in the overexpression mutants which became resistant to wide range of chemical fungicides. Moreover, P. digitatum knock-out mutants exhibited a lower rate of fungal virulence when infected oranges were stored at 20 °C. Disease symptoms were higher in the PdMFS1 overexpression mutants coming from the low-virulent P. digitatum parental strain. In addition, the gene expression analysis showed an induction of PdMFS1 transcription in all overexpression mutants regardless from which progenitor came from, and four-time intensification of the parental wild type strain during citrus infection reinforcing PdMFS1 role in fungal virulence. The P. digitatum MFS transporter PdMFS1 contributes not only to the acquisition of wide range of fungicide resistance but also in fungal virulence during citrus infection.
... In order to compare the various constructs and the different spin labels, all the EPR experiments were conducted at slightly alkaline pHs (7.2-7.5), under which the functional acidic residues are likely deprotonated 72 . Eight MdfA double cysteine variants were labeled in the periplasmic side with MTSSL, in the following referred to as NO, and C2-Gd, for a total of 16 constructs. ...
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.
... E. coli cells overexpressing MdfA exhibit MDR against numerous compounds, including cations such as tetraphenylphosphonium (TPP þ ) and electroneutral drugs such as chloramphenicol (Cm) (12). Protons and TPP þ ions bind to MdfA at different sites without, however, compromising the competitive nature of their binding (14). Previously, we reported the crystal structure of MdfA protein in its C in state (15). ...
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.
... The cycle is completed by reprotonation of carboxylates at the external side of the membrane with concomitant substrate release [15]. A similar model of transport has been proposed for MdfA [29]. ...
The secondary transporter LmrP from L. lactis is a remarkable model to study the molecular basis of secondary multidrug transport. This review article addresses more than twenty years of research about transport activity, substrates range, conformational dynamics and mechanistic models of drug export for LmrP. Several studies have shown that the transporter alternates between inward-open and outward-open conformations and that the transition is regulated by the protonation state of key acidic residues and is further modulated by the lipid environment.
... Biochemical and structural studies with EmrE, a small multidrug resistance H + -coupled multidrug antiporter, support a general mechanism for H + -coupled antiporters whereby the substrate and the protons cannot bind simultaneously to the protein (Schuldiner, 2014) and the overlap of the binding site results in a direct competition for its occupancy. In other examples, the "competition" seems to be indirect and is most likely achieved by some kind of allosteric mechanism (Fluman et al., 2012;Tanaka et al., 2013;Eicher et al., 2014;Schuldiner, 2014). To ensure the feasibility of such a mechanism, regardless of their specific structures or mechanisms, these antiporters have evolved so that they are exquisitely tuned to function at the very constant cytoplasmic pH maintained by cells. ...
The H ⁺ -coupled vesicular monoamine transporter (VMAT) is a transporter essential for life. VMAT mediates packaging of the monoamines serotonin, dopamine, norepinephrine, and histamine from the neuronal cytoplasm into presynaptic vesicles, which is a key step in the regulated release of neurotransmitters. However, a detailed understanding of the mechanism of VMAT function has been limited by the lack of availability of high-resolution structural data. In recent years, a series of studies guided by homology models has revealed significant insights into VMAT function, identifying residues that contribute to the binding site and to specific steps in the transport cycle. Moreover, to characterize the conformational transitions that occur upon binding of the substrate and coupling ion, we have taken advantage of the unique and powerful pharmacology of VMAT as well as of mutants that affect the conformational equilibrium of the protein and shift it toward defined conformations. This has allowed us to identify an important role for the proton gradient in driving a shift from lumen-facing to cytoplasm-facing conformations.
... In antiporters, substrates that are exchanged across the membrane usually compete with each other for a single binding site (Fluman et al., 2012), whereas in symporters, ion binding and substrate binding usually are coupled to be efficient (Jiang et al., 2013). In H þcoupled symporters, protonation was proposed to lower the energy barrier for substrate binding. ...
The human solute carriers (SLCs) comprise over 400 different transporters, organized into 65 families (http://slc.bioparadigms.org/) based on their sequence homology and transport function. SLCs are responsible for transporting extraordinarily diverse solutes across biological membranes, including inorganic ions, amino acids, lipids, sugars, neurotransmitters and drugs. Most of these membrane proteins function as coupled symporters (co-transporters) utilizing downhill ion (H⁺ or Na⁺) gradients as the driving force for the transport of substrate against its concentration gradient into cells. Other members work as antiporters (exchangers) that typically contain a single substrate-binding site with an alternating access mode of transport, while a few members exhibit channel-like properties. Dysfunction of SLCs is correlated with numerous human diseases and therefore they are potential therapeutic drug targets. In this review, we identified all of the SLC crystal structures that have been determined, most of which are from prokaryotic species. We further sorted all the SLC structures into four main groups with different protein folds and further discuss the well-characterized MFS (major facilitator superfamily) and LeuT (leucine transporter) folds. This review provides a systematic analysis of the structure, molecular basis of substrate recognition and mechanism of action in different SLC family members.
Pulsed dipolar spectroscopy, such as double electron-electron resonance (DEER), has been underutilized in protein structure determination, despite its ability to provide valuable spatial information. In this study, we present DEERefiner, a user-friendly MATLAB-based GUI program that enables the modeling of protein structures by combining an initial structure and DEER distance restraints. We illustrate the effectiveness of DEERefiner by successfully modeling the ligand-dependent conformational changes of the proton-drug antiporter LmrP to an extracellular-open-like conformation with an impressive precision of 0.76 Å. Additionally, DEERefiner was able to uncover a previously hypothesized but experimentally unresolved proton-dependent conformation of LmrP, characterized as an extracellular-closed/partially intracellular-open conformation, with a precision of 1.16 Å. Our work not only highlights the ability of DEER spectroscopy to model protein structures but also reveals the potential of DEERefiner to advance the field by providing an accessible and applicable tool for precise protein structure modeling, thereby paving the way for deeper insights into protein function.
Optimization of compound permeation into Gram-negative bacteria is one of the most challenging tasks in the development of antibacterial agents. Two permeability barriers─the passive diffusion barrier of the outer membrane (OM) and active drug efflux─act synergistically to protect cells from the antibacterial action of compounds. In Escherichia coli (E. coli) and relatives, these two barriers sieve compounds based on different physicochemical properties that are defined by their interactions with OM porins and efflux pumps, respectively. In this study, we critically tested the hypothesis that the best substrates and inhibitors of efflux pumps are compounds that can effectively permeate the OM and are available at relatively high concentrations in the periplasm. For this purpose, we filtered a large subset of the ZINC15 database of commercially available compounds for compounds containing a primary amine, a chemical feature known to facilitate the uptake through E. coli general porins. The assembled library was screened by ensemble docking to AcrA, the periplasmic component of the AcrAB-TolC efflux pump, followed by experimental testing of the top predicted binders for antibacterial activities, efflux recognition, and inhibition. We found that the filtered primary amine library is a rich source of compounds with efflux-inhibiting activities and identified efflux pump inhibitors with novel chemical scaffolds effective against E. coli AcrAB-TolC and efflux pumps of multidrug-resistant clinical isolates of Acinetobacter baumannii. However, primary amines are not required for the recognition of compounds by efflux pumps and their efflux-inhibitory activities.
Ineffectiveness of carbapenems against multidrug resistant pathogens led to the increased use of colistin (polymyxin E) as a last resort antibiotic. A gene belonging to the DedA family encoding conserved membrane proteins was previously identified by screening a transposon library of K. pneumoniae ST258 for sensitivity to colistin. We have renamed this gene dkcA ( d edA of K lebsiella required for c olistin resistance). DedA family proteins are likely membrane transporters required for viability of Escherichia coli and Burkholderia spp. at alkaline pH and for resistance to colistin in a number of bacterial species. Colistin resistance is often conferred via modification of the lipid A component of bacterial lipopolysaccharide with aminoarabinose (Ara4N) and/or phosphoethanolamine. Mass spectrometry analysis of lipid A of the ∆dkcA mutant shows a near absence of Ara4N in the lipid A, suggesting a requirement for DkcA for lipid A modification with Ara4N. Mutation of K. pneumoniae dkcA resulted in a reduction of the colistin minimal inhibitory concentration to approximately what is found with a Δ arnT strain. We also identify a requirement of DkcA for colistin resistance that is independent of lipid A modification, instead requiring maintenance of optimal membrane potential. K. pneumoniae Δ dkcA displays reduced virulence in Galleria mellonella suggesting colistin sensitivity can cause loss of virulence.
MdfA from Escherichia coli is a prototypical secondary multi-drug (Mdr) transporter that exchanges drugs for protons. MdfA mediated drug efflux is driven by the proton gradient and enabled by conformational changes, which accompany the recruitment of drugs and their release. In this work, we applied distance measurements by W-band double-electron electron resonance (DEER) spectroscopy to explore the binding of mito-TEMPO, a nitroxide-labeled substrate analog to Gd(III)-labeled MdfA. The choice of Gd(III)-nitroxide DEER enabled measurements in the presence of excess of mito-TEMPO, which has a relatively low affinity to MdfA. Distance measurements between mito-TEMPO and MdfA labeled at the periplasmic edges of either of three selected transmembrane helices (TM3¹⁰¹ ,TM5¹⁶⁸ and TM9³¹⁰) revealed rather similar distance distributions in detergent micelles (n-dodecyl-β-D-maltopyranoside )DDM)) and in lipid nanodiscs (ND). By grafting the predicted positions of the Gd(III) tag on the inward facing (If) crystal structure, we looked for binding positions which reproduced the maxima of the distance distributions. The results show that the location of the mito-TEMPO nitroxide in DDM-solubilized or ND-reconstituted MdfA is similar (only 0.4 nm apart). In both cases we located the nitroxide moiety near the ligand binding pocket in the If structure. However, according to the DEER derived position, the substrate clashes with TM11, suggesting that for mito-TEMPO-bound MdfA TM11 should move relative to the If structure. Additional DEER studies with MdfA labeled by Gd(III) at two sites revealed that TM9 also dislocates upon substrate binding. Together with our previous reports, this study demonstrates the utility of Gd(III)-Gd(III) and Gd(III)-nitroxide DEER measurements for studying the conformational behavior of transporters.
Cation and protons perform a substantial role in all the organism and its homeostasis within the cells are maintained by the cation-proton antiporters (CPAs). CPA is the huge family of the membrane transporter protein throughout the plant and animal kingdom including microorganism. In human, any malfunctioning of these proteins may lead to severe diseases like hypertension, heart diseases etc and CPAs are recently proposed to be responsible for the virulent property of various pathogens including Vibrio cholerae, Yersinia pestis etc. Human Sodium-Proton exchangers (Na⁺/H⁺ exchangers, NHEs) are crucial in ion homeostasis whereas Ec-NhaA, Na + -H + Antiporters maintain a balance of Na+ and proton in E. coli, regulating pH and cell volume within the cell. These Sodium-Proton antiporters are found to be responsible for the virulence in various pathogens causing human diseases. Understanding of these CPAs may assist investigators to target such human diseases, that further may lead to establishing the effective path for therapeutics or drug designing against associated human disease. Here we have compiled all such information on CPAs and provide a systematic approach to unravel the mechanism and role of antiporter proteins in a wide range of organisms. Being involved throughout all the species, this review on cation-proton antiporters may attract the attention of many investigators and concerned researchers and will be provided with the recent detailed information on the role of CPA in human health.
Members of the major facilitator superfamily of transporters (MFS) play an essential role in many physiological processes such as development, neurotransmission, and signaling. Aberrant functions of MFS proteins are associated with several diseases, including cancer, schizophrenia, epilepsy, amyotrophic lateral sclerosis and Alzheimer's disease. MFS transporters are also involved in multidrug resistance in bacteria and fungi. The structures of most MFS members, especially those of members with significant physiological relevance, are yet to be solved. The lack of structural and functional information impedes our detailed understanding, and thus the pharmacological targeting, of these transporters. To improve our knowledge on the mechanistic principles governing the function of MSF members, molecular dynamics (MD) simulations were performed on the inward-facing and outward-facing crystal structures of the human ferroportin homologue from the Gram-negative bacterium Bdellovibrio bacteriovorus (BdFpn). Several simulations with an excess of iron ions were also performed to explore the relationship between the protein's dynamics and the ligand recognition mechanism. The results reinforce the existence of the alternating-access mechanism already described for other MFS members. In addition, the reorganization of salt bridges, some of which are conserved in several MFS members, appears to be a key molecular event facilitating the conformational change of the transporter.
The prototypic multidrug (Mdr) transporter MdfA from E. coli efflux chemically-dissimilar substrates in exchange for protons. Similar to other transporters, MdfA purportedly functions by alternating access of a central substrate binding pocket to either side of the membrane. Accordingly, MdfA should open at the cytoplasmic side and/or laterally toward the membrane to enable access of drugs into its pocket. At the end of the cycle, the periplasmic side is expected to open to release drugs. Two distinct conformations of MdfA have been captured by X-ray crystallography: An outward open (Oo) conformation, stabilized by a Fab fragment, and a ligand-bound inward-facing (If) conformation, possibly stabilized by a mutation (Q131R). Here, we investigated how these structures relate to ligand-dependent conformational dynamics of MdfA in lipid bilayers. For this purpose, we combined distance measurements by Double Electron Electron Resonance (DEER) between pairs of spin labels in MdfA reconstituted in nanodiscs with cysteine cross-linking of natively expressed membrane-embedded MdfA variants. Our results suggest that in a membrane environment, MdfA assumes a relatively flexible, outward-closed/inward-closed (Oc/Ic) conformation. Unexpectedly, our data show that neither the substrate TPP nor protonation induces large scale conformational changes. Rather, we identified a substrate-responsive lateral gate which is open toward the inner leaflet of the membrane but closes upon drug binding. Together, our results suggest a modified model for the functional conformational cycle of MdfA that does not invoke canonical elements of alternating access.
Le biofouling est un phénomène invasif qui engendre des problèmes économiques et écologiques importants. Dans ce mode de vie privilégié, les micro-organismes communiquent grâce à un système de communication intra- et inter-espèces, voire inter-genre, appelé quorum sensing (QS). Le QS est basé sur les petites molécules diffusibles telles que les N-acyl homosérine lactones (AHL), et est donc impliqué dans de nombreux processus physiologiques, notamment la production de facteurs de virulence, l'émission de bioluminescence et la production de pigments. Dans une étude antérieure, le QS a été mis en évidence chez une bactérie marine dénommée Pseudoalteromonas ulvae TC14 isolée de la rade de Toulon. P. ulvae TC14 étant non seulement capable de produire un biofilm important et de la violacéine mais régulerait ces deux paramètres (biofilm et violacéine) par la détection d' AHLs exogènes car elle n'en produirait pas. Cette étude a consisté dans un premier temps à poursuivre la caractérisation du système QS chez P. ulvae TC 14 par une étude moléculaire. Tout en vérifiant l'absence de synthase d' AHL (Luxl) et des gènes potentiels de l'auto-induction de type 2 (AI-2) chez P. ulvae TC14, la présence de huit séquences régulatrices luxR a pu être détectée et une évaluation de l'expression de ces séquences a été faite par technique RT q-PCR. Dans le but de déterminer l'impact de la modulation du QS sur l'adhésion et la formation du biofilm chez P. ulvae TC 14 des molécules régulatrices du QS ont été testées en combinaison avec trois analogues synthétiques à effet antifouling. La restauration de l'adhésion et de la formation du biofilm par les molécules combinées a montré que la modulation du QS chez P. ulvae TC14 ne semble pas être la voie privilégiée pour l'inhibition du biofilm. Cependant l'évaluation d'autres cibles telles que la membrane bactérienne et les pompes d'efflux à travers des perméabilisants membranaires et les inhibiteurs de pompes d' efflux a montré un effet synergique sur l'adhésion et la formation du biofilm. Ainsi, la perméabilisation membranaire et l'inhibition des pompes d'efflux semblent être des meilleures voies de potentialisation de l'effet antifouling des analogues synthétiques
Objectives:
To identify major facilitator superfamily (MFS)-type chloramphenicol transporters of Acinetobacter baumannii AYE, to characterize its substrate specificity and identify CraA substrate and H+ binding sites.
Methods:
Five ORFs predicted to encode chloramphenicol transporters were heterologously expressed in Escherichia coli and their substrate specificity was determined by drug susceptibility assays on solid agar medium. CraA transport properties were determined via whole cell fluorescence experiments using ethidium and dequalinium. ACMA quenching was used to characterize the H+/drug antiport process in everted membrane vesicles. The function of CraA in A. baumannii was determined by drug susceptibility assay using A. baumannii ATCC 19606 ΔcraA.
Results:
CraA, ABAYE0913 and CmlA5 are functionally active when overproduced in E. coli. ABAYE0913 conferred resistance to florfenicol and benzalkonium, CmlA5 conferred resistance to chloramphenicol and thiamphenicol, and craA expression resulted in resistance to chloramphenicol, thiamphenicol, florfenicol, ethidium, dequalinium, chlorhexidine, benzalkonium, mitomycin C and TPP+. Cell expressing craA_E38A showed no resistance to all tested drugs, implying that Glu-38 is involved in the binding of drugs and/or protons. Functional assays indicated that substitution of Asp-46 to Ala resulted in severe susceptibility to cationic drugs, chloramphenicol and thiamphenicol. In contrast, Glu-338 is important for the recognition of chloramphenicol, florfenicol, chlorhexidine and dequalinium.
Conclusions:
This study suggests that CraA has a broad substrate specificity, similar to that of E. coli MdfA. However, due to the presence of three charged residues in the transmembrane region conferring different susceptibility profiles upon substitution to Ala, we postulate that CraA has a different substrate recognition mode compared with MdfA.
Active exchangers dissipate the gradient of one substrate to accumulate nutrients, export xenobiotics and maintain cellular homeostasis. Mechanistic studies have suggested that two fundamental properties are shared by all exchangers: substrate binding is antagonistic, and coupling is maintained by preventing shuttling of the empty transporter. The CLC H(+)/Cl(-) exchangers control the homeostasis of cellular compartments in most living organisms, but their transport mechanism remains unclear. We show that substrate binding to CLC-ec1 is synergistic rather than antagonistic: chloride binding induces protonation of a crucial glutamate. The simultaneous binding of H(+) and Cl(-) gives rise to a fully loaded state that is incompatible with conventional transport mechanisms. Mutations in the Cl(-) transport pathway identically alter the stoichiometries of H(+)/Cl(-) exchange and binding. We propose that the thermodynamics of synergistic substrate binding, rather than the kinetics of conformational changes and ion binding, determine the stoichiometry of transport.
The multidrug major facilitator superfamily transporter LmrP from Lactococcus lactis mediates protonmotive-force dependent efflux of amphiphilic ligands from the cell. We compared the role of membrane-embedded carboxylates in transport and binding of divalent cationic propidium and monovalent cationic ethidium. D235N, E327Q, and D142N replacements each resulted in loss of electrogenicity in the propidium efflux reaction, pointing to electrogenic 3H(+)/propidium(2+) antiport. During ethidium efflux, single D142N and D235N replacements resulted in apparent loss of electrogenicity, whereas the E327Q substitution did not affect the energetics, consistent with electrogenic 2H(+)/ethidium(+) antiport. Different roles of carboxylates were also observed in fluorescence anisotropy-based ligand-binding assays. Whereas D235 and E327 were both involved in propidium binding, the loss of one of these carboxylates could be compensated for by the other in ethidium binding. The D142N replacement did not affect the binding of either ligand. These data point to the presence of a dedicated proton binding site containing D142, and a flexible proton/ligand binding site containing D235 and E327, the contributions to proton and ligand binding of which depend on the chemical structure of the bound ligand. Our findings provide the first evidence that multidrug transport by secondary-active transporters can be associated with variable ion coupling.
Integral membrane proteins remain a challenge to proteomics because they contain domains with physicochemical properties poorly suited to today's bottom-up protocols. These transmembrane regions may potentially contain post-translational modifications of functional significance, and thus development of protocols for improved coverage in these domains is important. One way to achieve this goal is by using top-down mass spectrometry whereby the intact protein is subjected to mass spectrometry and dissociation. Here we describe top-down high resolution Fourier transform mass spectrometry with collisionally activated dissociation to study post-translationally modified integral membrane proteins with polyhelix bundle and transmembrane porin motifs and molecular masses up to 35 kDa. On-line LC-MS analysis of the bacteriorhodopsin holoprotein yielded b- and y-ions that covered the full sequence of the protein and cleaved 79 of 247 peptide bonds (32%). The experiment proved that the mature sequence consists of residues 14-261, confirming N-terminal propeptide cleavage and conversion of N-terminal Gln-14 to pyrrolidone carboxylic acid (-17.02 Da) and C-terminal removal of Asp-262. Collisionally activated dissociation fragments localized the N(6)-(retinylidene) modification (266.20 Da) between residues 225-248 at Lys-229, the sole available amine in this stretch. Off-line nanospray of all eight subunits of the cytochrome b(6)f complex from the cyanobacterium Nostoc PCC 7120 defined various post-translational modifications, including covalently attached c-hemes (615.17 Da) on cytochromes f and b. Analysis of murine mitochondrial voltage-dependent anion channel established the amenability of the transmembrane beta-barrel to top-down MS and localized a modification site of the inhibitor Ro 68-3400 at Cys-232. Where neutral loss of the modification is a factor, only product ions that carry the modification should be used to assign its position. Although bond cleavage in some transmembrane alpha-helical domains was efficient, other regions were refractory such that their primary structure could only be inferred from the coincidence of genomic translation with precursor and product ions that spanned them.
Multidrug (Mdr) transporters are membrane proteins that actively export structurally dissimilar drugs from the cell, thereby
rendering the cell resistant to toxic compounds. Similar to substrate-specific transporters, Mdr transporters also undergo
substrate-induced conformational changes. However, the mechanism by which a variety of dissimilar substrates are able to induce
similar transport-compatible conformational responses in a single transporter remains unclear. To address this major aspect
of Mdr transport, we studied the conformational behavior of the Escherichia coli Mdr transporter MdfA. Our results show that indeed, different substrates induce similar conformational changes in the transporter.
Intriguingly, in addition, we observed that compounds other than substrates are able to confer similar conformational changes
when covalently attached at the putative Mdr recognition pocket of MdfA. Taken together, the results suggest that the Mdr-binding
pocket of MdfA is conformationally sensitive. We speculate that the same conformational switch that usually drives active
transport is triggered promiscuously by merely occupying the Mdr-binding site.
Na(+)/H(+) antiporters are integral membrane proteins that exchange Na(+) for H(+) across the cytoplasmic membrane and many intracellular membranes. They are essential for Na(+), pH and volume homeostasis, which are crucial processes for cell viability. Accordingly, antiporters are important drug targets in humans and underlie salt-resistance in plants. Many Na(+)/H(+) antiporters are tightly regulated by pH. Escherichia coli NhaA Na(+)/H(+) antiporter, a prototype pH-regulated antiporter, exchanges 2 H(+) for 1 Na(+) (or Li(+)). The NhaA crystal structure has provided insights into the pH-regulated mechanism of antiporter action and opened up new in silico and in situ avenues of research. The monomer is the functional unit of NhaA yet the dimer is essential for the stability of the antiporter under extreme stress conditions. Ionizable residues of NhaA that strongly interact electrostatically are organized in a transmembrane fashion in accordance with the functional organization of the cation-binding site, ;pH sensor', the pH transduction pathway and the pH-induced conformational changes. Remarkably, NhaA contains an inverted topology motive of transmembrane segments, which are interrupted by extended mid-membrane chains that have since been found to vary in other ion-transport proteins. This novel structural fold creates a delicately balanced electrostatic environment in the middle of the membrane, which might be essential for ion binding and translocation. Based on the crystal structure of NhaA, a model structure of the human Na(+)/H(+) exchanger (NHE1) was constructed, paving the way to a rational drug design.
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.
The nature of the broad substrate specificity phenomenon, as manifested by multidrug resistance proteins, is not yet understood. In the Escherichia coli multidrug transporter, MdfA, the hydrophobicity profile and PhoA fusion analysis have so far identified only one membrane-embedded charged amino acid residue (E26). In order to determine whether this negatively charged residue may play a role in multidrug recognition, we evaluated the expression and function of MdfA constructs mutated at this position. Replacing E26 with the positively charged residue lysine abolished the multidrug resistance activity against positively charged drugs, but retained chloramphenicol efflux and resistance. In contrast, when the negative charge was preserved in a mutant with aspartate instead of E26, chloramphenicol recognition and transport were drastically inhibited; however, the mutant exhibited almost wild-type multidrug resistance activity against lipophilic cations. These results suggest that although the negative charge at position 26 is not essential for active transport, it dictates the multidrug resistance character of MdfA. We show that such a negative charge is also found in other drug resistance transporters, and its possible significance regarding multidrug resistance is discussed.
Highly active cytochrome b(6)f complexes from spinach and the cyanobacterium Mastigocladus laminosus have been analyzed by liquid chromatography with electrospray ionization mass spectrometry (LCMS+). Both size-exclusion and reverse-phase separations were used to separate protein subunits allowing measurement of their molecular masses to an accuracy exceeding 0.01% (+/-3 Da at 30,000 Da). The products of petA, petB, petC, petD, petG, petL, petM, and petN were detected in complexes from both spinach and M. laminosus, while the spinach complex also contained ferredoxin-NADP(+) oxidoreductase (Zhang, H., Whitelegge, J. P., and Cramer, W. A. (2001) Flavonucleotide:ferredoxin reductase is a subunit of the plant cytochrome b(6)f complex. J. Biol. Chem. 276, 38159-38165). While the measured masses of PetC and PetD (18935.8 and 17311.8 Da, respectively) from spinach are consistent with the published primary structure, the measured masses of cytochrome f (31934.7 Da, PetA) and cytochrome b (24886.9 Da, PetB) modestly deviate from values calculated based upon genomic sequence and known post-translational modifications. The low molecular weight protein subunits have been sequenced using tandem mass spectrometry (MSMS) without prior cleavage. Sequences derived from the MSMS spectra of these intact membrane proteins in the range of 3.2-4.2 kDa were compared with translations of genomic DNA sequence where available. Products of the spinach chloroplast genome, PetG, PetL, and PetN, all retained their initiating formylmethionine, while the nuclear encoded PetM was cleaved after import from the cytoplasm. While the sequences of PetG and PetN revealed no discrepancy with translations of the spinach chloroplast genome, Phe was detected at position 2 of PetL. The spinach chloroplast genome reports a codon for Ser at position 2 implying the presence of a DNA sequencing error or a previously undiscovered RNA editing event. Clearly, complete annotation of genomic data requires detailed expression measurements of primary structure by mass spectrometry. Full subunit coverage of an oligomeric intrinsic membrane protein complex by LCMS+ presents a new facet to intact mass proteomics.
The major facilitator superfamily represents the largest group of secondary membrane transporters in the cell. Here we report
the 3.3 angstrom resolution structure of a member of this superfamily, GlpT, which transports glycerol-3-phosphate into the
cytoplasm and inorganic phosphate into the periplasm. The amino- and carboxyl-terminal halves of the protein exhibit a pseudo
two-fold symmetry. Closed off to the periplasm, a centrally located substrate-translocation pore contains two arginines at
its closed end, which comprise the substrate-binding site. Upon substrate binding, the protein adopts a more compact conformation.
We propose that GlpT operates by a single–binding site, alternating-access mechanism through a rocker-switch type of movement.
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.
MdfA is an Escherichia coli multidrug-resistance transporter. Cells expressing MdfA from a multicopy plasmid exhibit multidrug resistance against a diverse group of toxic compounds. In this article, we show that, in addition to its role in multidrug resistance, MdfA confers extreme alkaline pH resistance and allows the growth of transformed cells under conditions that are close to those used normally by alkaliphiles (up to pH 10) by maintaining a physiological internal pH. MdfA-deleted E. coli cells are sensitive even to mild alkaline conditions, and the wild-type phenotype is restored fully by MdfA expressed from a plasmid. This activity of MdfA requires Na⁺ or K⁺. Fluorescence studies with inverted membrane vesicles demonstrate that MdfA catalyzes Na⁺- or K⁺-dependent proton transport, and experiments with reconstituted proteoliposomes confirm that MdfA is solely responsible for this phenomenon. Studies with multidrug resistance-defective MdfA mutants and competitive transport assays suggest that these activities of MdfA are related. Together, the results demonstrate that a single protein has an unprecedented capacity to turn E. coli from an obligatory neutrophile into an alkalitolerant bacterium, and they suggest a previously uncharacterized physiological role for MdfA in pH homeostasis.
• MdfA
• multidrug transport
• sodium proton antiporter
• alkaline pH tolerance
• Escherichia coli
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 largest family of solute transporters (major facilitator superfamily [MFS]) includes proton-motive-force-driven secondary
transporters. Several characterized MFS transporters utilize essential acidic residues that play a critical role in the energy-coupling
mechanism during transport. Surprisingly, we show here that no single acidic residue plays an irreplaceable role in the Escherichia coli secondary multidrug transporter MdfA.
The AcrA/AcrB/TolC complex spans the inner and outer membranes of Escherichia coli and serves as its major drug-resistance pump. Driven by the proton motive force, it mediates the efflux of bile salts, detergents,
organic solvents, and many structurally unrelated antibiotics. Here, we report a crystallographic structure of trimeric AcrB
determined at 2.9 and 3.0 angstrom resolution in space groups that allow asymmetry of the monomers. This structure reveals
three different monomer conformations representing consecutive states in a transport cycle. The structural data imply an alternating
access mechanism and a novel peristaltic mode of drug transport by this type of transporter.
MdfA is a 410-residue-long secondary multidrug transporter from E. coli. Cells expressing MdfA from a multicopy plasmid exhibit resistance against a diverse group of toxic compounds, including neutral and cationic ones, because of active multidrug export. As a prerequisite for high-resolution structural studies and a better understanding of the mechanism of substrate recognition and translocation by MdfA, we investigated its biochemical properties and overall structural characteristics. To this end, we purified the beta-dodecyl maltopyranoside (DDM)-solubilized protein using a 6-His tag and metal affinity chromatography, and size exclusion chromatography (SE-HPLC). Purified MdfA was analyzed for its DDM and phospholipid (PL) content, and tetraphenylphosphonium (TPP+)-binding activity. The results are consistent with MdfA being an active monomer in DDM solution. Furthermore, an investigation of two-dimensional crystals by electron crystallography and 3D reconstruction lent support to the notion that MdfA may also be monomeric in reconstituted proteoliposomes.
Cytoplasmic pH and periplasmic pH of Escherichia coli cells in suspension were observed with 4-s time resolution using fluorimetry of TorA-green fluorescent protein mutant 3* (TorA-GFPmut3*) and TetR-yellow fluorescent protein. Fluorescence intensity was correlated with pH using cell suspensions containing 20 mM benzoate, which equalizes the cytoplasmic pH with the external pH. When the external pH was lowered from pH 7.5 to 5.5, the cytoplasmic pH fell within 10 to 20 s to pH 5.6 to 6.5. Rapid recovery occurred until about 30 s after HCl addition and was followed by slower recovery over the next 5 min. As a control, KCl addition had no effect on fluorescence. In the presence of 5 to 10 mM acetate or benzoate, recovery from external acidification was diminished. Addition of benzoate at pH 7.0 resulted in cytoplasmic acidification with only slow recovery. Periplasmic pH was observed using TorA-GFPmut3* exported to the periplasm through the Tat system. The periplasmic location of the fusion protein was confirmed by the observation that osmotic shock greatly decreased the periplasmic fluorescence signal by loss of the protein but had no effect on the fluorescence of the cytoplasmic protein. Based on GFPmut3* fluorescence, the pH of the periplasm equaled the external pH under all conditions tested, including rapid acid shift. Benzoate addition had no effect on periplasmic pH. The cytoplasmic pH of E. coli was measured with 4-s time resolution using a method that can be applied to any strain construct, and the periplasmic pH was measured directly for the first time.
EmrE is an Escherichia coli H⁺-coupled multidrug transporter that provides a unique experimental paradigm because of its small size and stability, and because its activity can be studied in detergent solution. In this work, we report a study of the transient kinetics of substrate binding and substrate-induced proton release in EmrE. For this purpose, we measured transient changes in the tryptophan fluorescence upon substrate binding and the rates of substrate-induced proton release. The fluorescence of the essential and fully conserved Trp residue at position 63 is sensitive to the occupancy of the binding site with either protons or substrate. The maximal rate of binding to detergent-solubilized EmrE of TPP⁺, a high-affinity substrate, is 2 × 10⁷ M⁻¹·s⁻¹, a rate typical of diffusion-limited reactions. Rate measurements with medium- and low-affinity substrates imply that the affinity is determined mainly by the k off of the substrate. The rates of substrate binding and substrate-induced release of protons are faster at basic pHs and slower at lower pHs. These findings imply that the substrate-binding rates are determined by the generation of the species capable of binding; this is controlled by the high affinity to protons of the glutamate at position 14, because an Asp replacement with a lower pK is faster at the same pHs.
• drug resistance
• fluorescence
• ion-coupled transporter
• membrane protein
• transient kinetic
The Escherichia coli multidrug transporter MdfA contains a membrane-embedded charged residue (Glu-26) that was shown to play an important role in substrate recognition. To identify additional determinants of multidrug recognition we isolated 58 intragenic second-site mutations that restored the function of inactive MdfA E26X mutants. In addition, two single-site mutations that enhanced the activity of wild-type MdfA were identified. Most of the mutations were found in two regions, the cytoplasmic half of transmembrane segments (TMs) 4, 5, and 6 (cluster 1) and the periplasmic half of TM 1 and 2 (cluster 2). The identified residues were mutated to cysteines in the background of a functional cysteine-less MdfA, and substrate protection against alkylation was analyzed. The results support the suggestion that the two clusters are involved in substrate recognition. Using inverted membrane vesicles we observed that a proton electrochemical gradient (Deltamicro(H(+)), inside positive and acidic) enhanced the substrate-protective effect in the cytoplasmic region, whereas it largely reduced this effect in the periplasmic side of MdfA. Therefore, we propose that substrates interact with two sites in MdfA, one in the cytoplasmic leaflet of the membrane and the other in the periplasmic leaflet. Theoretically, these domains could constitute a large part of the multidrug pathway through MdfA.
The major facilitator superfamily (MFS) represents the largest group of secondary active membrane transporters, and its members transport a diverse range of substrates. Recent work shows that MFS antiporters, and perhaps all members of the MFS, share the same three-dimensional structure, consisting of two domains that surround a substrate translocation pore. The advent of crystal structures of three MFS antiporters sheds light on their fundamental mechanism; they operate via a single binding site, alternating-access mechanism that involves a rocker-switch type movement of the two halves of the protein. In the sn-glycerol-3-phosphate transporter (GlpT) from Escherichia coli, the substrate-binding site is formed by several charged residues and a histidine that can be protonated. Salt-bridge formation and breakage are involved in the conformational changes of the protein during transport. In this review, we attempt to give an account of a set of mechanistic principles that characterize all MFS antiporters.
The major facilitator superfamily (MFS) transporters are an ancient and widespread family of secondary active transporters. In Escherichia coli, the uptake of l-fucose, a source of carbon for microorganisms, is mediated by an MFS proton symporter, FucP. Despite intensive study of the MFS transporters, atomic structure information is only available on three proteins and the outward-open conformation has yet to be captured. Here we report the crystal structure of FucP at 3.1 Å resolution, which shows that it contains an outward-open, amphipathic cavity. The similarly folded amino and carboxyl domains of FucP have contrasting surface features along the transport path, with negative electrostatic potential on the N domain and hydrophobic surface on the C domain. FucP only contains two acidic residues along the transport path, Asp 46 and Glu 135, which can undergo cycles of protonation and deprotonation. Their essential role in active transport is supported by both in vivo and in vitro experiments. Structure-based biochemical analyses provide insights into energy coupling, substrate recognition and the transport mechanism of FucP.
A remarkably high pKa of approximately 10.5 has been determined for sugar-binding affinity to the lactose permease of Escherichia coli (LacY), indicating that, under physiological conditions, substrate binds to fully protonated LacY. We have now systematically tested site-directed replacements for the residues involved in sugar binding, as well as H+ translocation and coupling, in order to determine which residues may be responsible for this alkaline pKa. Mutations in the sugar-binding site (Glu126, Trp151, Glu269) markedly decrease affinity for sugar but do not alter the pKa for binding. In contrast, replacements for residues involved in H+ translocation (Arg302, Tyr236, His322, Asp240, Glu325, Lys319) exhibit pKa values for sugar binding that are either shifted toward neutral pH or independent of pH. Values for the apparent dissociation constant for sugar binding (K(d)(app)) increase greatly for all mutants except neutral replacements for Glu325 or Lys319, which are characterized by remarkably high affinity sugar binding (i.e., low K(d)(app)) from pH 5.5 to pH 11. The pH dependence of the on- and off-rate constants for sugar binding measured directly by stopped-flow fluorometry implicates k(off) as a major factor for the affinity change at alkaline pH and confirms the effects of pH on K(d)(app) inferred from steady-state fluorometry. These results indicate that the high pKa for sugar binding by wild-type LacY cannot be ascribed to any single amino acid residue but appears to reside within a complex of residues involved in H+ translocation. There is structural evidence for water bound in this complex, and the water could be the site of protonation responsible for the pH dependence of sugar binding.
The three-component AcrA/AcrB/TolC efflux system of Escherichia coli catalyzes the proton motive force-driven extrusion of a variety of cytotoxic compounds. The inner membrane pump component AcrB belongs to the resistance nodulation and cell division (RND) superfamily and is responsible for drug specificity and energy transduction of the entire tripartite efflux system. Systematic mutational analysis of titratable and polar membrane-located amino acids revealed four residues, D407, D408, K940, and, R971, to be of prime importance for AcrB function. Using matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, D408 was shown to specifically react with dicyclohexylcarbodiimide (DCCD) in a pH-dependent manner. The apparent pK(a) of D408 of 7.4 would enable binding and release of protons under physiological conditions. In contrast to other secondary transporters, D408 was not protected from carbodiimide modification in the presence of drugs, which supports the notion of spatially separated transport pathways for drugs and protons. This study provides evidence for a substantial role of membrane-located carboxylates as a central element of the proton translocation pathway in AcrB and other members of the RND superfamily.
Multidrug transporters are membrane proteins that expel a wide spectrum of cytotoxic compounds from the cell. Through this function, they render cells resistant to multiple drugs. These transporters are found in many different families of transport proteins, of which the largest is the major facilitator superfamily. Multidrug transporters from this family are highly represented in bacteria and studies of them have provided important insight into the mechanism underlying multidrug transport. This review summarizes the work carried out on these interesting proteins and underscores the differences and similarities to other transport systems.
Classical neurotransmitters are transported into synaptic vesicles so that their release can be regulated by neural activity. In addition, the vesicular transport of biogenic amines modulates susceptibility to N-methyl-4-phenylpyridinium (MPP+), the active metabolite of the neurotoxin N-methyl-1,2,3,6-tetrahydropyridine that produces a model of Parkinson's disease. Taking advantage of selection in MPP+, we have used gene transfer followed by plasmid rescue to identify a cDNA clone that encodes a vesicular amine transporter. The sequence predicts a novel mammalian protein with 12 transmembrane domains and homology to a class of bacterial drug resistance transporters. We have detected messenger RNA transcripts for this transporter only in the adrenal gland. Monoamine cell populations in the brain stem express a distinct but highly related protein.
This review discusses recent advances in our understanding of the structure, function and molecular genetics of the membrane domain of red cell anion exchanger, band 3 (AE1), and its role in red cell and kidney disease. A new model for the topology of band 3 has been proposed, which suggests the membrane domain has 12 membrane spans, rather than the 14 membrane spans of earlier models. The major difference between the models is in the topology of the region on the C-terminal side of membrane spans 1-7. Two dimensional crystals of the deglycosylated membrane domain of band 3 have yielded two and three dimensional projection maps of the membrane domain dimer at low resolution. The human band 3 gene has been completely sequenced and this has facilitated the study of natural band 3 mutations and their involvement in disease. About 20% of hereditary spherocytosis cases arise from heterozygosity for band 3 mutations, and result in the absence or decrease of the mutant protein in the red cell membrane. Several other natural band 3 mutations are known that appear to be clinically benign, but alter red cell phenotype or are associated with altered red cell blood group antigens. These include the mutant band 3 present in Southeast Asian ovalocytosis, a condition which provides protection against cerebral malaria in children. Familial distal renal tubular acidosis, a condition associated with kidney stones, has been shown to result from a novel group of band 3 mutations. The total absence of band 3 has been described in animals-occurring naturally in cattle and after targeted disruption in mice. Some of these severely anaemic animals survive, so band 3 is not strictly essential for life. Although the band 3-negative red cells were very unstable, they contained a normally-assembled red cell skeleton, suggesting that the bilayer of the normal red cell membrane is stabilized by band 3 interactions with membrane lipids, rather than by interactions with the spectrin skeleton.
Integration of biochemical and biophysical data on the lactose permease of Escherichia coli has culminated in a molecular model that predicts substrate-protein proximities which include interaction of a hydroxyl group in the galactopyranosyl ring with Glu269. In order to test this hypothesis, we studied covalent modification of carboxyl groups with carbodiimides using electrospray ionization mass spectrometry (ESI-MS) and demonstrate that substrate protects the permease against carbodiimide reactivity. Further more, a significant proportion of the decrease in carbodiimide reactivity occurs specifically in a nanopeptide containing Glu269. In contrast, carbodiimide reactivity of mutant Glu269-->Asp that exhibits lower affinity is unaffected by substrate. By monitoring the ability of different substrate analogs to protect against carbodiimide modification of Glu269, it is suggested that the C-3 OH group of the galactopyranosyl ring may play an important role in specificity, possibly by H-bonding with Glu269. The approach demonstrates that mass spectrometry can provide a powerful means of analyzing ligand interactions with integral membrane proteins.
Membrane transport proteins that transduce free energy stored in electrochemical ion gradients into a concentration gradient are a major class of membrane proteins. We report the crystal structure at 3.5 angstroms of the Escherichia coli lactose permease, an intensively studied member of the major facilitator superfamily of transporters. The molecule is composed of N- and C-terminal domains, each with six transmembrane helices, symmetrically positioned within the permease. A large internal hydrophilic cavity open to the cytoplasmic side represents the inward-facing conformation of the transporter. The structure with a bound lactose homolog, beta-D-galactopyranosyl-1-thio-beta-D-galactopyranoside, reveals the sugar-binding site in the cavity, and residues that play major roles in substrate recognition and proton translocation are identified. We propose a possible mechanism for lactose/proton symport (co-transport) consistent with both the structure and a large body of experimental data.
According to the current topology model of the Escherichia coli multidrug transporter MdfA, it contains a membrane-embedded negatively charged residue, Glu26, which was shown to play an important role in substrate recognition. To further elucidate the role of this substrate recognition determinant, various Glu26 replacements were characterized. Surprisingly, studies with neutral MdfA substrates showed that, unlike many enzymatic systems where the size and chemical properties of binding site residues are relatively defined, MdfA tolerates a variety of changes at position 26, including size, hydrophobicity, and charge. Moreover, although efficient transport of positively charged substrates requires a negative charge at position 26 (Glu or Asp), neutralization of this charge does not always abrogate the interaction of MdfA with cationic drugs, thus demonstrating that the negative charge does not play an essential role in the multidrug transport mechanism. Collectively, these results suggest a link between the broad substrate specificity profile of multidrug transporters and the structural and chemical promiscuity at their substrate recognition pockets.
Drug and multidrug resistance have greatly compromised the compounds that were once the mainstays of antibiotic therapy. This resistance often persists despite reductions in the use of antibiotics, indicating that the proteins encoded by antibiotic-resistance genes have alternative physiological roles that can foster such persistence in the absence of selective pressure by antibiotics. The recent observations that Tet(L), a tetracycline-efflux transporter, and MdfA, a multidrug-efflux transporter, both confer alkali tolerance offer a striking case study in support of this hypothesis.
A very fast empirical method is presented for structure-based protein pKa prediction and rationalization. The desolvation effects and intra-protein interactions, which cause variations in pKa values of protein ionizable groups, are empirically related to the positions and chemical nature of the groups proximate to the pKa sites. A computer program is written to automatically predict pKa values based on these empirical relationships within a couple of seconds. Unusual pKa values at buried active sites, which are among the most interesting protein pKa values, are predicted very well with the empirical method. A test on 233 carboxyl, 12 cysteine, 45 histidine, and 24 lysine pKa values in various proteins shows a root-mean-square deviation (RMSD) of 0.89 from experimental values. Removal of the 29 pKa values that are upper or lower limits results in an RMSD = 0.79 for the remaining 285 pKa values.
MdfA is an Escherichia coli multidrug transporter of the major facilitator superfamily (MFS) of secondary transporters. Although several aspects of multidrug recognition by MdfA have been characterized, better understanding the detailed mechanism of its function requires structural information. Previous studies have modeled the 3D structures of MFS proteins, based on the X-ray structure of LacY and GlpT. However, because of poor sequence homology, between LacY, GlpT, and MdfA additional constraints were required for a reliable homology modeling. Using an algorithm that predicts the angular orientation of each transmembrane helix (TM) (kPROT), we obtained a remarkably similar pattern for the 12 TMs of MdfA and those of GlpT and LacY, suggesting that they all have similar helix packing. Consequently, a 3D model was constructed for MdfA by structural alignment with LacY and GlpT, using the kPROT results as an additional constraint. Further refinement and a preliminary evaluation of the model were achieved by correlated mutation analysis and the available experimental data. Surprisingly, in addition to the previously characterized membrane-embedded glutamate at position 26, the model suggests that Asp34 and Arg112 are located within the membrane, on the same face of the cavity as Glu26. Importantly, Arg112 is evolutionarily conserved in secondary drug transporters, and here we show that a positive charge at this position is absolutely essential for multidrug transport by MdfA.
The steps in the mechanism of proton transport in bacteriorhodopsin include examples for most kinds of proton transfer reactions that might occur in a transmembrane pump: proton transfer via a bridging water molecule, coupled protonation/deprotonation of two buried groups separated by a considerable distance, long-range proton migration over a hydrogen-bonded aqueous chain, and capture as well as release of protons at the membrane-water interface. The conceptual and technical advantages of this system have allowed close examination of many of these model reactions, some at an atomic level.
Chronic kidney disease (CKD) has been linked to higher stroke risk. Anemia is a common consequence of CKD, and recent evidence suggests anemia may increase risk of cardiovascular events. The combined effect of CKD and anemia on stroke risk, however, has not been investigated thoroughly. We analyzed data from a middle-aged, community-based cohort to determine if CKD and anemia interacted to affect stroke risk.
Data on 13,716 participants in the prospective Atherosclerosis Risk in Communities (ARIC) Study were analyzed to assess the joint effect of CKD and anemia on risk of incident stroke during a 9-year follow-up period. CKD was defined as a creatinine clearance of <60 mL/min. Anemia was defined as hemoglobin levels of <13 g/dL for men or <12 g/dL for women.
Overall, CKD was associated with an increase in stroke risk after adjustment for other factors [hazard ratio HR) 1.81; 95% CI 1.26 to 2.02]. However, this association was modified substantially by anemia. In the presence of anemia, CKD was associated with a substantially higher risk of stroke compared to no CKD (HR 5.43; 95% CI 2.04 to 14.41). In contrast, when anemia was not present, CKD was associated with only a modest, nonsignificant elevation in stroke risk (HR 1.41; 95% CI 0.93 to 2.14). The interaction between CKD and anemia on risk of stroke was statistically significant (P < 0.01).
Among middle-aged, community-based persons, the combination of CKD and anemia was associated with a substantial increase in stroke risk, independent of other known risk factors for stroke.
Top-down mass spectrometry focuses on intact proteins, thereby avoiding loss of information accompanying 'shotgun' protocols that reduce the proteome to a collection of peptides. A suite of liquid-chromatography technologies has been developed for purification of intact integral membrane proteins in aqueous/organic solvent mixtures compatible with biological 'soft-ionization' mass spectrometry, preserving covalent structure into the gas phase. Multiply charged protein ions are fragmented in the gas phase, using either collision-activated or electron-capture dissociation, thus yielding complex spectra of sequence-dependent product ions that collectively define the original native covalent state of an intact protein. Top down offers a more detail-orientated approach to post-transcriptional and post-translational diversity allowing an enhanced insight beyond genomic translation, which has now extended into the bilayer proteome.
LmrP is a secondary active multidrug transporter from Lactococcus lactis. The protein belongs to the major facilitator superfamily and utilizes the electrochemical proton gradient (inside negative and alkaline) to extrude a wide range of lipophilic cations from the cell. Previous work has indicated that ethidium, a monovalent cationic substrate, is exported by LmrP by electrogenic antiport with two (or more) protons. This observation raised the question whether these protons are translocated sequentially along the same pathway, or through different routes. To address this question, we constructed a 3-D homology model of LmrP based on the high-resolution structure of the glycerol-3P/Pi antiporter GlpT from Escherichia coli, and we tested by mutagenesis the possible proton conduction points suggested by this model. Similar to the template, LmrP is predicted to contain an internal cavity formed at the interface between the two halves of the transporter. On the surface of this cavity lie two clusters of polar, aromatic and carboxylate residues with potentially important function in proton shuttling. Cluster 1 in the C-terminal half contains D235 and E327 in immediate proximity of each other, and is located near the apex of the cavity. Cluster 2 in the N-terminal half contains D142. Analyses of LmrP mutants containing charge-conservative or carboxyl-to-amide replacements at positions 142, 235 and 327 suggest that D142 is part of a dedicated proton translocation pathway in the ethidium translocation reaction. In contrast, D235 and E327 are part of an independent pathway, in which D235 interacts with protons. E327 appears to modulate the pKa of D235 and plays a role in the interaction with ethidium. These results are consistent with the proposal that major facilitator superfamily proteins consist of two membrane domains, one of which is involved in substrate binding and the other in ion coupling, and they indicate that there are two proton conduction pathways at play in the transport mechanism.
The effect of bulk-phase pH on the apparent affinity (K dapp) of purified wild-type lactose permease (LacY) for sugars was studied. K dapp values were determined by ligand-induced changes in the fluorescence of either of two covalently bound fluorescent reporters positioned away from the sugar-binding site. K dapp for three different galactopyranosides was determined over a pH range from 5.5 to 11. A remarkably high pKa of ≈10.5 was obtained for all sugars. Kinetic data for thiodigalactoside binding measured from pH 6 to 10 show that decreased affinity for sugar at alkaline pH is due specifically to increased reverse rate. A similar effect was also observed with nitrophenylgalactoside by using a direct binding assay. Because affinity for sugar remains constant from pH 5.5 to pH 9.0, it follows that LacY is fully protonated with respect to sugar binding under physiological conditions of pH. The results are consistent with the conclusion that LacY is protonated before sugar binding during lactose/H⁺ symport in either direction across the membrane.
• lactose permease
• membrane transporters
• pH titrations
• proton translocation
• substrate affinity