Functional reconstitution and osmoregulatory properties of the ProU ABC transporter from Escherichia coli

Abstract

The ATP-binding cassette (ABC) transporter ProU from Escherichia coli translocates a wide range of compatible solutes and contributes to the regulation of cell volume, which is particularly important when the osmolality of the environment fluctuates. We have purified the components of ProU, i.e., the substrate-binding protein ProX, the nucleotide-binding protein ProV and the transmembrane protein ProW, and reconstituted the full transporter complex in liposomes. We engineered a lipid anchor to ProX for surface tethering of this protein to ProVW-containing proteoliposomes. We show that glycine betaine binds to ProX with high-affinity and is transported via ProXVW in an ATP-dependent manner. The activity ProU is salt and anionic lipid-dependent and mimics the ionic strength-gating of transport of the homologous OpuA system.     

Nucleotide dependent monomer/dimer equilibrium of OpuAA, the nucleotide-binding protein of the osmotically regulated ABC transporter OpuA from Bacillus subtilis

The OpuA system of Bacillus subtilis is a member of the substrate-binding-protein-dependent ABC transporter superfamily and serves for the uptake of the compatible solute glycine betaine under hyperosmotic growth conditions. Here, we have characterized the nucleotide-binding protein (OpuAA) of the B.subtilis OpuA transporter in vitro. OpuAA was overexpressed heterologously in Escherichia coli as a hexahistidine tag fusion protein and purified to homogeneity by affinity and size exclusion chromatography (SEC). Dynamic monomer/dimer equilibrium was observed for OpuAA, and the K(D) value was determined to be 6 microM. Under high ionic strength assay conditions, the monomer/dimer interconversion was diminished, which enabled separation of both species by SEC and separate analysis of both monomeric and dimeric OpuAA. In the presence of 1 M NaCl, monomeric OpuAA showed a basal ATPase activity (K(M)=0.45 mM; k(2)=2.3 min(-1)), whereas dimeric OpuAA showed little ATPase activity under this condition. The addition of nucleotides influenced the monomer/dimer ratio of OpuAA, demonstrating different oligomeric states during its catalytic cycle. The monomer was the preferred species under post-hydrolysis conditions (e.g. ADP/Mg(2+)), whereas the dimer dominated the nucleotide-free and ATP-bound states. The affinity and stoichiometry of monomeric or dimeric OpuAA/ATP complexes were determined by means of the fluorescent ATP-analog TNP-ATP. One molecule of TNP-ATP was bound in the monomeric state and two TNP-ATP molecules were detected in the dimeric state of OpuAA. Binding of TNP-ADP/Mg(2+) to dimeric OpuAA induced a conformational change that led to the decay of the dimer. On the basis of our data, we propose a model that couples changes in the oligomeric state of OpuAA with ATP hydrolysis.     

Immunization with the basic membrane protein (BMP) family ABC transporter elicits protection against Enterococcus faecium in a murine infection model

Enterococcus faecium is evolving as a multi-resistant pathogen causing infections with high morbidity and mortality. A protective vaccine against E. faecium is lacking up till now. ATP-binding cassette (ABC) transporter proteins have important functions in bacteria to maintain survival and homeostasis. In the present study, we evaluated the basic membrane protein (BMP) family ABC transporter substrate-binding protein, designated herein as BMP, as a potential vaccine candidate against E. faecium. Recombinant BMP of E. faecium was expressed in Escherichia coli, and purified by metal affinity chromatography. Swiss albino mice were immunized with the recombinant BMP combined with Bacillus Calmette–Guérin (BCG) and/or alum as adjuvants. Mice immunized with BMP combined with alternating BCG and alum developed BMP-specific IgG and were protected against E. faecium challenge as evidenced from organ bioburden and histopathological examination. Furthermore, serum from immunized mice showed enhanced opsonophagocytic activity and protected mice against E. faecium challenge by passive immunization. Bioinformatic analysis revealed appreciable degrees of homology between E. faecium BMP and proteins from other pathogens which suggests BMP could be a useful vaccine against multiple pathogens. To our knowledge, this is the first report of in-vivo evaluation of BMP as a potential vaccine candidate against E. faecium.     

The cydD gene product, component of a heterodimeric ABC transporter, is required for assembly of periplasmic cytochrome c and of cytochrome bd in Escherichia coli

Abstract The cydD gene of Escherichia coli encodes a protein which, together with the CydC protein, probably constitutes a heterodimeric, ABC-family membrane transporter, necessary for biosynthesis of the cytochrome bd quinol oxidase. Here, we demonstrate that a cydD mutant also fails to synthesise periplasmic c -type cytochrome(s), suggesting that the transporter exports haem or some other component involved in assembly of cytochromes that are found in, or exposed to, the periplasm. The CydDC system appears to be the first example of a transporter required for periplasmic cytochrome assembly processes requiring more than one type of haem. A mutant defective in trxB (adjacent to the cydDC operon, and encoding thioredoxin reductase) was unaffected in cytochrome c or bd assembly.     

A Step-by-step Method for the Reconstitution of an ABC Transporter into Nanodisc Lipid Particles

The nanodisc is a discoidal particle (~ 10-12 nm large) that trap membrane proteins into a small patch of phospholipid bilayer. The nanodisc is a particularly attractive option for studying membrane proteins, especially in the context of ligand-receptor interactions. The method pioneered by Sligar and colleagues is based on the amphipathic properties of an engineered highly a-helical scaffold protein derived from the apolipoprotein A1. The hydrophobic faces of the scaffold protein interact with the fatty acyl side-chains of the lipid bilayer whereas the polar regions face the aqueous environment. Analyses of membrane proteins in nanodiscs have significant advantages over liposome because the particles are small, homogeneous and water-soluble. In addition, biochemical and biophysical methods normally reserved to soluble proteins can be applied, and from either side of the membrane. In this visual protocol, we present a step-by-step reconstitution of a well characterized bacterial ABC transporter, the MalE-MalFGK₂ complex. The formation of the disc is a self-assembly process that depends on hydrophobic interactions taking place during the progressive removal of the detergent. We describe the essential steps and we highlight the importance of choosing a correct protein-to-lipid ratio in order to limit the formation of aggregates and larger polydisperse liposome-like particles. Simple quality controls such as gel filtration chromatography, native gel electrophoresis and dynamic light scattering spectroscopy ensure that the discs have been properly reconstituted.     

Engineered zinc-binding sites confirm proximity and orientation of transmembrane helices I and III in the human serotonin transporter

The human serotonin transporter (hSERT) regulates neurotransmission by removing released serotonin (5-HT) from the synapse. Previous studies identified residues in SERT transmembrane helices (TMHs) I and III as interaction sites for substrates and antagonists. Despite an abundance of data supporting a 12-TMH topology, the arrangement of the TMHs in SERT and other biogenic amine transporters remains undetermined. A high-resolution structure of a bacterial leucine transporter that demonstrates homology with SERT has been reported, thus providing the basis for the development of a SERT model. Zn2+-binding sites have been utilized in transporters and receptors to define experimentally TMH proximity. Focusing on residues near the extracellular ends of hSERT TMHs I and III, we engineered potential Zn2+-binding sites between V102 or W103 (TMH I) and I179-L184 (TMH III). Residues were mutated to either histidine or cysteine. TMH I/III double mutants were constructed from functional TMH I mutants, and Zn2+ sensitivity was assessed. Dose-response assays suggest an approximately twofold increase in sensitivity to Zn2+ inhibition at the hSERT V102C/M180C and approximately fourfold at the V102C/I179C mutant compared to the hSERT V102C single mutant. We propose that the increased sensitivity to Zn2+ confirms the proximity and the orientation of TMHs I and III in the membrane. Homology modeling of the proposed Zn2+-binding sites using the coordinates of the Aquifex aeolicus leucine transporter structure provided a structural basis for interpreting the results and developing conclusions.     

Membrane Topology and Functional Importance of the Periplasmic Region of ABC Transporter LolCDE

The LolCDE complex is an ATP-binding cassette transporter that mediates the release of newly synthesized lipoproteins from the cytoplasmic membrane of gram-negative bacteria, which results in the initiation of outer-membrane sorting of lipoproteins through the Lol pathway. LolCDE is composed of one copy each of membrane subunits LolC and LolE, and two copies of nucleotide-binding subunit LolD. In this study, we examined the membrane topology of LolC and LolE by PhoA fusion analysis. Both LolC and LolE were found to have four transmembrane segments with a large periplasmic loop exposed to the periplasm. Despite similarities in sequence and topology, the accessibility of a sulfhydryl reagent to Cys introduced into the periplasmic loop suggested that the structure of the periplasmic region differs between LolC and LolE. Inhibition of the release of lipoproteins by the sulfhydryl reagent supported a previous proposal that LolC and LolE have distinct functions.     

An important role of a "probable ATP-binding component of ABC transporter" during the process of Pseudomonas aeruginosa resistance to fluoroquinolone

In order to find new drug target to eliminate the fluoroquinolone resistance, the in vitro progress of Pseudomonas aeruginosa fluoroquinolone resistance was mimicked, and then proteomic analysis was applied to comparing different protein profiles during the resistant process. The results show that the expression of a "probable ATP-binding component of ATP binding cassette (ABC) transporter" existed in ciprofloxacin-intermediate and -resistant strains, but not in sensitive strain. In addition, the ciprofloxacin concentrations in P. aeruginosa strains, which were obtained from the progress of P. aeruginosa fluoroquinolone resistance, were determined by means of HPLC; the results show that the decrease of the intracellular concentration of drug and the expression of this new protein nearly take place simultaneously. The changes of mRNA levels of the probable ATP-binding component of ABC transporter were detected by virtue of RT-PCR and showed that this protein did not express in the sensitive strains but expressed increasingly in the intermediate and resistant strains. In order to determine the relationships between the development of antibiotic resistance and this protein further, a DNAzyme was designed to aim at the mRNA of the probable ATP-binding component of ABC transporter directly; the ciprofloxacin resistance of P. aeruginosa was partially reduced in vivo by inhibiting the expression of this protein. This DNAzyme has no effect on sensitive strain. And the comparison of drug intracellular concentrations between DNAzyme-treated strains and its control strains shows that this protein may be included in the course of active drug efflux.     

Managing the manganese: molecular mechanisms of manganese transport and homeostasis

Manganese (Mn) is an essential metal nutrient for plants. Recently, some of the genes responsible for transition metal transport in plants have been identified; however, only relatively recently have Mn2+ transport pathways begun to be identified at the molecular level. These include transporters responsible for Mn accumulation into the cell and release from various organelles, and for active sequestration into endomembrane compartments, particularly the vacuole and the endoplasmic reticulum. Several transporter gene families have been implicated in Mn2+ transport, including cation/H+ antiporters, natural resistance-associated macrophage protein (Nramp) transporters, zinc-regulated transporter/iron-regulated transporter (ZRT/IRT1)-related protein (ZIP) transporters, the cation diffusion facilitator (CDF) transporter family, and P-type ATPases. The identification of mutants with altered Mn phenotypes can allow the identification of novel components in Mn homeostasis. In addition, the characterization of Mn hyperaccumulator plants can increase our understanding of how plants can adapt to excess Mn, and ultimately allow the identification of genes that confer this stress tolerance. The identification of genes responsible for Mn2+ transport has substantially improved our understanding of plant Mn homeostasis.     

Sequence analysis and functional studies of a chromosomal region of alkaliphilic Bacillus firmus OF4 encoding an ABC-type transporter with similarity of sequence and Na+ exclusion capacity to the Bacillus subtilis NatAB transporter

A 14.1-kb DNA fragment was cloned from a lambda library containing inserts of DNA from alkaliphilic Bacillus firmus OF4 on the basis of its hybridization to a probe from a previously sequenced alkaliphile homolog of the natA gene from Bacillus subtilis. Sequence analysis of the entire fragment revealed that, as in B. subtilis, the natA gene was part of a putative gene locus encoding an ABC-type transporter. In the alkaliphile, the transporter involved three genes, designated natCAB, that are part of a larger operon of unknown function. This is in contrast to the two-gene natAB operon and to another homolog from B. subtilis, the yhaQP genes. Like natAB, however, the alkaliphile natCAB catalyzes Na⁺ extrusion as assessed in a mutant of Escherichia coli that is deficient in Na⁺ extrusion. The full 14.1-kb fragment of alkaliphile DNA sequenced in this study contained several probable operons as well as likely monocistronic units. Among the 17 predicted ORFs apart from natCAB were acsA, a homolog of a halobacterial gene encoding acetylCoA synthetase; sspA, a homolog of a small acid-soluble spore protein; and malK, an ATP-binding component that was unaccompanied by candidates for other mal transport genes but was able to complement a malK-deficient mutant of E. coli. No strong candidates for genes encoding a secondary Na⁺/H⁺ antiporter were found in the fragment, either from the sequence analysis or from analyses of complementation of E. coli mutants by subclones of the 14.1-kb piece. There were a total of 12 ORFs whose closest and significant homologs were genes from B. subtilis; of these, one-third were in apparently different contexts, as assessed by the sequence of the neighboring genes, than the B. subtilis homologs. Received: August 30, 1998 / Accepted: November 13, 1998     

The first N-terminal transmembrane helix of each subunit of the antigenic peptide transporter TAP is essential for independent tapasin binding

The heterodimeric ABC transporter TAP translocates proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum, where these peptides are loaded onto MHC class I molecules by a macromolecular peptide-loading complex (PLC) and subsequently shuttled to the cell surface for inspection by cytotoxic T lymphocytes. Tapasin recruits, as a central adapter protein, other components of the PLC at the unique N-terminal domains of TAP. We found that the N-terminal domains of human TAP1 and TAP2 can independently bind to tapasin, thus providing two separate loading platforms for PLC assembly. Moreover, tapasin binding is dependent on the first N-terminal transmembrane helix of TAP1 and TAP2, demonstrating that these two helices contribute independently to the recruitment of tapasin and associated factors.     

Mutation of a novel ABC transporter gene is responsible for the failure to incorporate uric acid in the epidermis of ok mutants of the silkworm,Bombyx mori

ok mutants of the silkworm, Bombyx mori, exhibit highly translucent larval skin resulting from the inability to incorporate uric acid into the epidermal cells. Here we report the identification of a gene responsible for the ok mutation using positional cloning and RNAi experiments. In two independent ok mutant strains, we found a 49-bp deletion and a 233-bp duplication, respectively, in mRNAs of a novel gene, Bm-ok, which encodes a half-type ABC transporter, each of which results in translation of a truncated protein in each mutant. Although the Bm-ok sequence was homologous to well-known transporter genes, white, scarlet, and brown in Drosophila, the discovery of novel orthologs in the genomes of lepidopteran, hymenopteran, and hemipteran insects identifies it as a member of a new distinct subfamily of transporters. Embryonic RNAi of Bm-ok demonstrated that repression of Bm-ok causes a translucent phenotype in the first-instar silkworm larva. We discuss the possibility that Bm-ok forms a heterodimer with another half-type ABC transporter, Bmwh3, and acts as a uric acid transporter in the silkworm epidermis.     

Tyrosine-728 and glutamic acid-735 are essential for the metalloproteolytic activity of the lethal factor of Bacillus anthracis

The lethal factor (LF) of Bacillus anthracis is a Zn2+-endopeptidase specific for the MAPK-kinase family of proteins. The catalytic zinc atom is coordinated by a first shell of residues including the two histidines and the glutamate of the zinc-binding motif HExxH and by Glu-735. A characteristic feature of LF is the presence, within the second shell of residues, of a tyrosine (Tyr-728) in close proximity (3.3 A) to the zinc atom. To investigate the role of Tyr-728 and Glu-735, LF mutants with one or both of these two residues replaced by Ala were cloned, expressed, and purified from Escherichia coli. A fourth mutant was obtained by replacing Tyr-728 with Phe. Spectroscopic analysis of these mutants indicates that they fold in the same way as the parental molecule and that zinc stabilizes the structure of LF. These mutants have neither proteolytic activity nor in vivo toxicity. The possible role of Tyr-728 in catalysis is discussed.     

Iron Complexes and Their Reactivity in the Bleomycin Assay for Radical-Promoting Loosely-Bound Iron

The sensitivity of the bleomycin assay for loosely-bound iron depends on the concentration of bleomycin and ascorbic acid and the pH of the reaction. The non-haem-iron proteins transferrin, conalbumin and ferritin release iron at an acid pH value, whereas the haem-iron proteins release iron more readily at an alkaline pH. In addition, haem proteins are liable to release iron when peroxides are present. Organic peroxides and hydrogen peroxide can be produced during the bleomycin reaction leading to iron release from haem proteins. However, this can be prevented from reacting with bleomycin by adding zinc ions to the reaction following addition of the sample. Iron already bound to bleomycin is not displaced by zinc whereas zinc bound to bleomycin is not displaced by iron allowing 'free' and 'released' iron to be discriminated.     

Sequence,biophysical, and structural analyses of the PstS lipoprotein (BB0215) from Borrelia burgdorferi reveal a likely binding component of an ABC‐type phosphate transporter

The Lyme disease agent Borrelia burgdorferi, which is transmitted via a tick vector, is dependent on its tick and mammalian hosts for a number of essential nutrients. Like other bacterial diderms, it must transport these biochemicals from the extracellular milieu across two membranes, ultimately to the B. burgdorferi cytoplasm. In the current study, we established that a gene cluster comprising genes bb0215 through bb0218 is cotranscribed and is therefore an operon. Sequence analysis of these proteins suggested that they are the components of an ABC‐type transporter responsible for translocating phosphate anions from the B. burgdorferi periplasm to the cytoplasm. Biophysical experiments established that the putative ligand‐binding protein of this system, BbPstS (BB0215), binds to phosphate in solution. We determined the high‐resolution (1.3 Å) crystal structure of the protein in the absence of phosphate, revealing that the protein's fold is similar to other phosphate‐binding proteins, and residues that are implicated in phosphate binding in other such proteins are conserved in BbPstS. Taken together, the gene products of bb0215‐0218 function as a phosphate transporter for B. burgdorferi.     

Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin

Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that ⁷⁷⁰DYWL⁷⁷³ of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around ⁷⁷⁰DYWL⁷⁷³ of ECL 4 in the ABCC2.     

Conserved residues in the HIV-1 Nef hydrophobic pocket are essential for recruitment and activation of the Hck tyrosine kinase

The Nef protein of the primate lentiviruses human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is essential for high-titer viral replication and acquired immune deficiency syndrome (AIDS) progression. Nef binds to the macrophage-specific Src family member Hck through its SH3 domain, resulting in constitutive kinase activation capable of transforming rodent fibroblasts. Nef-Hck interaction may be essential for M-tropic HIV replication and AIDS pathogenesis, identifying this virus-host protein complex as a rational target for anti-HIV drug discovery. Here, we investigated whether interaction with Hck is a common feature of Nef alleles from different strains of HIV-1. We compared the ability of four different laboratory HIV-1 Nef alleles (SF2, LAI, ELI, and Consensus) to induce Hck activation and transformation in our Rat-2 fibroblast model. While SF2, LAI, and Consensus Nef all bound and activated Hck, ELI Nef failed to bind to the Hck SH3 domain in vitro and did not cooperate with Hck in fibroblast transformation. Molecular modeling identified three residues in the core region of SF2 Nef (Ala83, His116, and Tyr120) which are substituted in ELI with Glu, Asn, and Ile, respectively. Two of these residues (Ala83 and Tyr120) form part of the hydrophobic pocket that contacts Ile 96 in the RT loop of the Hck SH3 domain in the Nef-SH3 crystal structure. Substitution of SF2 Nef Tyr120 with Ile completely abolished Hck recruitment and activation. In a complementary experiment, substitution of ELI Ile120 with Tyr partly restored ELI Nef-induced Hck activation and transformation in Rat-2 cells. Hck activation increased further by substitution of ELI Glu83 with Ala and Asn116 with His, suggestive of a supportive role for these residues in Hck binding. This study provides the first biological evidence that the HIV-1 Nef hydrophobic pocket is critical to Hck recruitment and activation in vivo. Targeting the Nef hydrophobic pocket with a small molecule may be sufficient to disrupt Nef signaling through Hck in HIV-infected macrophages, slowing disease progression.     

An enhanced expression of ABC 1 transporter in circulating leukocytes as a potential molecular marker for the diagnostics of glaucoma

Summary. Objective: Glaucoma is a neurodegenerative disease. Since vascular dysregulation is supposed to be a risk factor for the development of glaucomatous damage, the preventive treatment might slow down the disease development. The efficiency of the therapeutic treatment depends particularly on a drug efflux pump regulated by ABC transporters. ABC 1 is also known to participate on the vascular regulation. This study was focused on the comparative analysis of ABC 1 expression levels in circulating leukocytes of non-glaucomatous individuals and glaucoma patients.Results and conclusions: The expression rates of ABC 1 were significantly increased in leukocytes of glaucoma patients compared to non-glaucomatous individuals. The expression level of ABC 1 was, furthermore, highly homogeneous in glaucoma patients. In contrast, these expression levels in non-glaucomatous individuals were extremely heterogeneous. This transporter acts as the energy-dependent unidirectional transmembrane cholesterol efflux pump and can export a wide range of hydrophobic drugs. Additionally an observed enhanced ABC 1 expression in circulating leukocytes may be implicated in the vascular regulation mechanisms of glaucoma. We proposed the enhanced expression of ABC 1 in leukocytes as a potential marker for the diagnostics and ex vivo molecular monitoring of glaucoma.     

The Thr505 and Ser557 residues of the AGT1-encoded alpha-glucoside transporter are critical for maltotriose transport in Saccharomyces cerevisiae

Aims:  The main objective of this study was to identify amino acid residues in the AGT1‐encoded α‐glucoside transporter (Agt1p) that are critical for efficient transport of maltotriose in the yeast Saccharomyces cerevisiae. Methods and Results:  The sequences of two AGT1‐encoded α‐glucoside transporters with different efficiencies of maltotriose transport in two Saccharomyces strains (WH310 and WH314) were compared. The sequence variations and discrepancies between these two proteins (Agt1p_WH310 and Agt1p_WH314) were investigated for potential effects on the functionality and maltotriose transport efficiency of these two AGT1‐encoded α‐glucoside transporters. A 23‐amino‐acid C‐terminal truncation proved not to be critical for maltotriose affinity. The identification of three amino acid differences, which potentially could have been instrumental in the transportation of maltotriose, were further investigated. Single mutations were created to restore the point mutations I505T, V549A and T557S one by one. The single site mutant V549A showed a decrease in maltotriose transport ability, and the I505T and T557S mutants showed complete reduction in maltotriose transport. Conclusions:  The amino acids Thr⁵⁰⁵ and Ser⁵⁵⁷, which are respectively located in the transmembrane (TM) segment TM¹¹ and on the intracellular segment after TM¹² of the AGT1‐encoded α‐glucoside transporters, are critical for efficient transport of maltotriose in S. cerevisiae. Significance and Impact of the Study:  Improved fermentation of starch and its dextrin products, such as maltotriose and maltose, would benefit the brewing and whisky industries. This study could facilitate the development of engineered maltotriose transporters adapted to starch‐efficient fermentation systems, and offers prospects for the development of yeast strains with improved maltose and maltotriose uptake capabilities that, in turn, could increase the overall fermentation efficiencies in the beer and whisky industries.