smFRET Probing Reveals Substrate-Dependent Conformational Dynamics of E. coli Multidrug MdfA

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.     

A New Critical Conformational Determinant of Multidrug Efflux by an MFS Transporter

Secondary multidrug (Mdr) transporters utilize ion concentration gradients to actively remove antibiotics and other toxic compounds from cells. The model Mdr transporter MdfA from Escherichia coli exchanges dissimilar drugs for protons. The transporter should open at the cytoplasmic side to enable access of drugs into the Mdr recognition pocket. Here we show that the cytoplasmic rim around the Mdr recognition pocket represents a previously overlooked important regulatory determinant in MdfA. We demonstrate that increasing the positive charge of the electrically asymmetric rim dramatically inhibits MdfA activity and sometimes even leads to influx of planar, positively charged compounds, resulting in drug sensitivity. Our results suggest that unlike the mutants with the electrically modified rim, the membrane-embedded wild-type MdfA exhibits a significant probability of an inward-closed conformation, which is further increased by drug binding. Since MdfA binds drugs from its inward-facing environment, these results are intriguing and raise the possibility that the transporter has a sensitive, drug-induced conformational switch, which favors an inward-closed state.     

Structure,Dynamics, and Substrate-induced Conformational Changes of the Multidrug Transporter EmrE in Liposomes

EmrE, a member of the small multidrug transporters superfamily, extrudes positively charged hydrophobic compounds out of Escherichia coli cytoplasm in exchange for inward movement of protons down their electrochemical gradient. Although its transport mechanism has been thoroughly characterized, the structural basis of energy coupling and the conformational cycle mediating transport have yet to be elucidated. In this study, EmrE structure in liposomes and the substrate-induced conformational changes were investigated by systematic spin labeling and EPR analysis. Spin label mobilities and accessibilities describe a highly dynamic ligand-free (apo) conformation. Dipolar coupling between spin labels across the dimer reveals at least two spin label populations arising from different packing interfaces of the EmrE dimer. One population is consistent with antiparallel arrangement of the monomers, although the EPR parameters suggest deviations from the crystal structure of substrate-bound EmrE. Resolving these discrepancies requires an unusual disposition of TM3 relative to the membrane-water interface and a kink in its backbone that enables bending of its C-terminal part. Binding of the substrate tetraphenylphosphonium changes the environment of spin labels and their proximity in three transmembrane helices. The underlying conformational transition involves repacking of TM1, tilting of TM2, and changes in the backbone configurations of TM3 and the adjacent loop connecting it to TM4. A dynamic apo conformation is necessary for the polyspecificity of EmrE allowing the binding of structurally diverse substrates. The flexibility of TM3 may play a critical role in movement of substrates across the membrane.     

A Multidrug ABC Transporter with a Taste for Salt

Background

LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H⁺-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known.

Methodology/Principal Findings

We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H⁺-Na⁺-Cl⁻ symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift.

Conclusions/Significance

The observations on H⁺-Na⁺-Cl⁻ co-transport substantiate earlier suggestions of H⁺-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter.     

The Remarkable Transport Mechanism of P-Glycoprotein: A Multidrug Transporter

Human P-glycoprotein (ABCB1) is a primary multidrug transporter located in plasma membranes, that utilizes the energy of ATP hydrolysis to pump toxic xenobiotics out of cells. P-glycoprotein employs a most unusual molecular mechanism to perform this drug transport function. Here we review our work to elucidate the molecular mechanism of drug transport by P-glycoprotein. High level heterologous expression of human P-glycoprotein, in the yeast Saccharomyces cerevisiae, has facilitated biophysical studies in purified proteoliposome preparations. Development of novel spin-labeled transport substrates has allowed for quantitative and rigorous measurements of drug transport in real time by EPR spectroscopy. We have developed a new drug transport model of P-glycoprotein from the results of mutagenic, quantitative thermodynamic and kinetic studies. This model satisfactorily accounts for most of the unusual kinetic, coupling, and physiological features of P-glycoprotein. Additionally, an atomic detail structural model of P-glycoprotein has been devised to place our results within a proper structural context.     

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

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

Transported Substrate Determines Exchange Rate in the Multidrug Resistance Transporter EmrE

EmrE, a small multidrug resistance transporter, serves as an ideal model to study coupling between multidrug recognition and protein function. EmrE has a single small binding pocket that must accommodate the full range of diverse substrates recognized by this transporter. We have studied a series of tetrahedral compounds, as well as several planar substrates, to examine multidrug recognition and transport by EmrE. Here we show that even within this limited series, the rate of interconversion between the inward- and outward-facing states of EmrE varies over 3 orders of magnitude. Thus, the identity of the bound substrate controls the rate of this critical step in the transport process. The binding affinity also varies over a similar range and is correlated with substrate hydrophobicity within the tetrahedral substrate series. Substrate identity influences both the ground-state and transition-state energies for the conformational exchange process, highlighting the coupling between substrate binding and transport required for alternating access antiport.     

Structural Basis of Drug Recognition by the Multidrug Transporter ABCG2

ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E₁S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.     

Dissecting the Conformational Dynamics of the Bile Acid Transporter Homologue ASBTNM

Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na⁺ as the driving force. The crystal structures of two bacterial homologues ASBT_NM and ASBT_Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na⁺-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na⁺ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT_NM under different Na⁺ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT_NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na⁺ ions shift the conformational equilibrium of ASBT_NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters.     

A Conformational Switch in the Scaffolding Protein NHERF1 Controls Autoinhibition and Complex Formation

The mammalian Na⁺/H⁺ exchange regulatory factor 1 (NHERF1) is a multidomain scaffolding protein essential for regulating the intracellular trafficking and macromolecular assembly of transmembrane ion channels and receptors. NHERF1 consists of tandem PDZ-1, PDZ-2 domains that interact with the cytoplasmic domains of membrane proteins and a C-terminal (CT) domain that binds the membrane-cytoskeleton linker protein ezrin. NHERF1 is held in an autoinhibited state through intramolecular interactions between PDZ2 and the CT domain that also includes a C-terminal PDZ-binding motif (-SNL). We have determined the structures of the isolated and tandem PDZ2CT domains by high resolution NMR using small angle x-ray scattering as constraints. The PDZ2CT structure shows weak intramolecular interactions between the largely disordered CT domain and the PDZ ligand binding site. The structure reveals a novel helix-turn-helix subdomain that is allosterically coupled to the putative PDZ2 domain by a network of hydrophobic interactions. This helical subdomain increases both the stability and the binding affinity of the extended PDZ structure. Using NMR and small angle neutron scattering for joint structure refinement, we demonstrate the release of intramolecular domain-domain interactions in PDZ2CT upon binding to ezrin. Based on the structural information, we show that human disease-causing mutations in PDZ2, R153Q and E225K, have significantly reduced protein stability. Loss of NHERF1 expressed in cells could result in failure to assemble membrane complexes that are important for normal physiological functions.     

Asymmetric Conformational Flexibility in the ATP-Binding Cassette Transporter HI1470/1

Putative metal-chelate-type ABC transporter HI1470/1 is homologous with vitamin B₁₂ importer BtuCD but exhibits a distinct inward-facing conformation in contrast to the outward-facing conformation of BtuCD. Normal-mode analysis of HI1470/1 reveals the intrinsic asymmetric conformational flexibility in this transporter and demonstrates that the transition from the inward-facing to the outward-facing conformation is realized through the asymmetric motion of individual subunits of the transporter. This analysis suggests that the asymmetric arrangement of the BtuC dimer in the crystal structure of the BtuCD-F complex represents an intermediate state relating HI1470/1 and BtuCD. Furthermore, a twisting motion between transmembrane domains and nucleotide-binding domains encoded in the lowest-frequency normal mode of this type of importer is found to contribute to the conformational transitions during the whole cycle of substrate transportation. A more complete translocation mechanism of the BtuCD type importer is proposed.     

Benzodiazepine-Mediated Structural Changes in the Multidrug Transporter P-Glycoprotein: An Intrinsic Fluorescence Quenching Analysis

P-glycoprotein expressed in Pichia pastoris was used to study the drug binding sites of different benzodiazepines. The effect of bromazepam, chlordiazepoxide, diazepam and flurazepam on P-glycoprotein structure was investigated by measuring the intrinsic fluorescence of the transporter tryptophan residues. Purified mouse mdr1a transporter in mixed micelles of 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonic acid and 1,2-dimiristoyl-sn-glycerol-3-phosphocholine emitted fluorescence at 340 nm indicative of the fluorophores in a relatively apolar environment. Acrylamide and iodide ion were used as collisional quenchers toward distinct regions of the transporter, the protein and the interface protein-surface, respectively. Binding of ATP induced conformational changes at the protein surface level in accordance with the location of the nucleotide binding sites. Bromazepam interaction with the transporter was located at the protein-surface interface, diazepam at the membrane region and chlordiazepoxide at the protein surface. Only the flurazepam interaction site was not detected by the quenchers used. All benzodiazepines were able to elicit reorientation of the protein fluorophores on the P-glycoprotein—ATP complex.     

In vitro Unfolding and Refolding of the Small Multidrug Transporter EmrE

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

Effects of Multidrug Resistance-Related ATP-Binding-Cassette Transporter Proteins on the Cytoskeletal Activity of Cytochalasins

Cytochalasins are microfilament-active mould metabolites, widely utilized to study the involvement of the actin cytoskeleton in cellular processes as well as in genotoxicity and cell kinetic research. In this study we have investigated whether multidrug-resistance phenotypes, caused by overexpression of the ATP-binding-cassette transporter proteins P-glycoprotein (P-gp) or multidrug-resistance-associated protein (MRP), influence the microfilament-depolymerizing effect of cytochalasins. Using four well-characterized multidrug-resistance cell models, we have shown that both the microfilament-disrupting (phalloidine staining) and the cytotoxic (MTT-assay) activity of cytochalasins are reduced in parallel with increased P-gp expression and restorable by P-gp-modulating agents. This also applied to the cytochalasin D-mediated induction of polykaryons (microscopic evaluation) which arise as a consequence of impaired cytokinesis but unaffected karyokinesis. The reduced cellular activity of cytochalasins in P-gp-positive cell lines was correlated with decreased intracellular accumulation ([³H]cytochalasin B accumulation) which was also restorable by P-gp modulators. Moreover, the dose-dependent inhibition of P-gp photoaffinity labeling ([³H]azidopine) suggested cytochalasins as P-gp-binding agents. In contrast, MRP overexpression had no effect on either cytochalasin microfilament activity or cytotoxicity. In conclusion, data indicate that the microfilament-destructive effects of cytochalasins are impaired due to a reduction of the intracellular cytochalasin accumulation by P-gp but not by MRP. Results are discussed with regard to P-gp as a resistance factor when cytochalasins are utilized to study microfilament dynamics, cell cycle kinetics or chromosomal damage. Moreover, the polykaryon-inducing activity of cytochalasin D is suggested as a specific indicator for a P-gp-mediated multidrug-resistance phenotype and the reversing potency of chemosensitizers.     

Conformational Switch and Role of Phosphorylation in PAK Activation

p21-activated protein kinases (PAKs) are involved in signal transduction processes initiating a variety of biological responses. They become activated by interaction with Rho-type small GTP-binding proteins Rac and Cdc42 in the GTP-bound conformation, thereby relieving the inhibition of the regulatory domain (RD) on the catalytic domain (CD). Here we report on the mechanism of activation and show that proteolytic digestion of PAK produces a heterodimeric RD-CD complex consisting of a regulatory fragment (residues 57 to 200) and a catalytic fragment (residues 201 to 491), which is active in the absence of Cdc42. Cdc42-GppNHp binds with low affinity (K(d) 0.6 microM) to intact kinase, whereas the affinity to the isolated regulatory fragment is much higher (K(d) 18 nM), suggesting that the difference in binding energy is used for the conformational change leading to activation. The full-length kinase, the isolated RD, and surprisingly also their complexes with Cdc42 behave as dimers on a gel filtration column. Cdc42-GppNHp interaction with the RD-CD complex is also of low affinity and does not dissociate the RD from the CD. After autophosphorylation of the kinase domain, Cdc42 binds with high (14 nM) affinity and dissociates the RD-CD complex. Assuming that the RD-CD complex mimics the interaction in native PAK, this indicates that the small G protein may not simply release the RD from the CD. It acts in a more subtle allosteric control mechanism to induce autophosphorylation, which in turn induces the release of the RD and thus full activation.