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Bile acids perform vital functions in the human liver and are the essential component of bile. It is therefore not surprising that the biology of bile acids is extremely complex, regulated on different levels, and involves soluble and membrane receptors as well as transporters. Hereditary disorders of these proteins manifest in different pathophysiological processes that result in liver diseases of varying severity. In this review, we summarize our current knowledge of the physiology and pathophysiology of bile acids with an emphasis on recently established analytical approaches as well as the molecular mechanisms that underlie signaling and transport of bile acids. In this review, we will focus on ABC transporters of the canalicular membrane and their associated diseases. As the G protein-coupled receptor, TGR5, receives increasing attention, we have included aspects of this receptor and its interaction with bile acids.

ABC transporters (ATP-binding cassette transporter) traffic drugs and their metabolites across membranes, making ABC transporter expression levels a key factor regulating local drug concentrations in different tissues and individuals. Yet, quantification of ABC transporters remains challenging because they are large and low-abundance transmembrane proteins. Here, we analysed 200 samples of crude and membrane-enriched fractions from human liver, kidney, intestine, brain microvessels and skin, by label-free quantitative mass spectrometry. We identified 32 (out of 48) ABC transporters: ABCD3 was the most abundant in liver, whereas ABCA8, ABCB2/TAP1 and ABCE1 were detected in all tissues. Interestingly, this atlas unveiled that ABCB2/TAP1 may have TAP2-independent functions in the brain and that biliary atresia (BA) and control livers have quite different ABC transporter profiles. We propose that meaningful biological information can be derived from a direct comparison of these data sets.

ATP-binding cassette (ABC) transporters are fascinating molecular machines that are capable of transporting a large variety of chemically diverse compounds. The energy required for translocation is derived from binding and hydrolysis of ATP. All ABC transporters share a basic architecture and are composed of two transmembrane domains and two nucleotide binding domains (NBDs). The latter harbor all conserved sequence motifs that hallmark the ABC transporter superfamily. The NBDs form the nucleotide binding sites (NBSs) in their interface. Transporters with two active NBSs are called canonical transporters, while ABC exporters from eukaryotic organisms, including humans, frequently have a degenerate NBS1 containing noncanonical residues that strongly impair ATP hydrolysis. Here, we summarize current knowledge on degenerate ABC transporters. By integrating structural information with biophysical and biochemical evidence of asymmetric function, we develop a model for the transport cycle of degenerate ABC transporters. We will elaborate on the unclear functional advantages of a degenerate NBS.

Bacteria produce under certain stress conditions bacteriocins and microcins that display antibacterial activity against closely related species for survival. Bacteriocins and microcins exert their antibacterial activity by either disrupting the membrane or inhibiting essential intracellular processes of the bacterial target. To this end, they can lyse bacterial membranes and cause subsequent loss of their integrity or nutrients, or hijack membrane receptors for internalisation. Both bacteriocins and microcins are ribosomally synthesised and several are posttranslationally modified, whereas others are not. Such peptides are also toxic to the producer bacteria, which utilise immunity proteins or/and dedicated ATP-binding cassette (ABC) transporters to achieve self-immunity and peptide export. In this review, we discuss the structure and mechanism of self-protection that is conferred by these ABC transporters.

ABC transporters are ubiquitously present in all kingdoms and mediate the transport of a large spectrum of structurally different compounds. Plants possess high numbers of ABC transporters in relation to other eukaryotes; the ABCG subfamily in particular is extensive. Earlier studies demonstrated that ABCG transporters are involved in important processes influencing plant fitness. This review summarizes the functions of ABCG transporters present in the model plant Arabidopsis thaliana. These transporters take part in diverse processes such as pathogen response, diffusion barrier formation, or phytohormone transport. Studies involving knockout mutations reported pleiotropic phenotypes of the mutants. In some cases, different physiological roles were assigned to the same protein. The actual transported substrate(s), however, still remain to be determined for the majority of ABCG transporters. Additionally, the proposed substrate spectrum of different ABCG proteins is not always reflected by sequence identities between ABCG members. Applying only reverse genetics is thereby insufficient to clearly identify the substrate(s). We therefore stress the importance of in vitro studies in addition to in vivo studies in order to (i) clarify the substrate identity; (ii) determine the transport characteristics including directionality; and (iii) identify dimerization partners of the half-size proteins, which might in turn affect substrate specificity.

Secretion systems are essential for Gram-negative bacteria as these nanomachineries allow a communication with the outside world by exporting proteins into the extracellular space or directly into the cytosol of a host cell. For example, type one secretion systems (T1SS) secrete a broad range of substrates across both membranes into the extracellular space. One well-known example is the hemolysin A (HlyA) T1SS from Escherichia coli (E. coli), which consists of an ABC transporter (HlyB), a membrane fusion protein (HlyD), the outer membrane protein TolC and the substrate HlyA, a member of the family of RTX (repeats in toxins) toxins. Here, we determined the amount of TolC at the endogenous level (parental strain, UTI89) and under conditions of overexpression (T7 expression system, BL21(DE3)-BD). The overall amount of TolC was not influenced by the overexpression of the HlyBD complex. Moving one step further, we determined the localization of the HlyA T1SS by super-resolution microscopy. In contrast to other bacterial secretion systems, no polarization was observed with respect to endogenous or overexpression levels. Additionally, the cell growth and division cycle did not influence the polarization. Most importantly, the size of the observed T1SS clusters did not correlate with the recently proposed outer membrane islands. These data indicate that T1SS cluster at the outer membrane generating domains of so far not described identity. Importance Uropathogenic Escherichia coli (UPEC) strains cause about 110 million urinary tract infections each year worldwide representing a global burden to the healthcare system. UPEC secrete many virulence factors among these the TX toxin hemolysin A via a cognate T1SS into the extracellular space. In this study, we determined the endogenous copy number of the HlyA T1SS in UTI89 and analyzed the surface localization in BL21(DE3)-BD and UTI89, respectively. With approximately 800 copies of the T1SS in UTI89, this is one of the highest expressed bacterial secretion systems. Furthermore and in clear contrast to other secretion systems, no polarized surface localization was detected. Finally, quantitative analysis of the super-resolution data revealed that clusters of the HlyA T1SS are not related to the recently identified outer membrane protein islands. These data provide insights into the quantitative molecular architecture of the HlyA T1SS.

The ABC transporter Pdr5 of S. cerevisiae is a key player of the PDR network that works as a first line of defense against a wide range of xenobiotic compounds. As the first discovered member of the family of asymmetric PDR ABC transporters, extensive studies have been carried out to elucidate the molecular mechanism of drug efflux and the details of the catalytic cycle. Pdr5 turned out to be an excellent model system to study functional and structural characteristics of asymmetric, uncoupled ABC transporters. However, to date studies have been limited to in vivo or plasma membrane systems, as it was not possible to isolate Pdr5 in a functional state. Here, we describe the solubilization and purification of Pdr5 to homogeneity in a functional state as confirmed by in vitro assays. The ATPase deficient Pdr5 E1036Q mutant was used as a control and proves that detergent-purified wild-type Pdr5 is functional resembling in its activity the one in its physiological environment. Finally, we show that the isolated active Pdr5 is monomeric in solution. Taken together, our results described in this study will enable a variety of functional investigations on Pdr5 required to determine molecular mechanism of this asymmetric ABC transporter.

Several ABC transporters of the human liver are responsible for the secretion of bile salts, lipids and cholesterol. Their interplay protects the biliary tree from the harsh detergent activity of bile salts. Among these transporters, ABCB4 is essential for the translocation of phosphatidylcholine (PC) lipids from the inner to the outer leaflet of the canalicular membrane of hepatocytes. ABCB4 deficiency can result in altered PC to bile salt ratios, which led to intrahepatic cholestasis of pregnancy, low phospholipid associated cholelithiasis, drug induced liver injury or even progressive familial intrahepatic cholestasis type 3. Although PC lipids only account for 30–40% of the lipids in the canalicular membrane, 95% of all phospholipids in bile are PC lipids. We discuss this discrepancy in the light of PC synthesis and bile salts favoring certain lipids. Nevertheless, the in vivo extraction of PC lipids from the outer leaflet of the canalicular membrane by bile salts should be considered as a separate step in bile formation. Therefore, methods to characterize disease causing ABCB4 mutations should be considered carefully, but such an analysis represents a crucial point in understanding the currently unknown transport mechanism of this ABC transporter.

The two major efflux pump systems are involved in multidrug resistance (MDR): (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ABC transporters that are involved in MDR and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of transport and specificity remain elusive. Here, we provide electrophysiological data on reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Importantly, a strictly substrate dependent co-transport of protons was also observed in in vitro transport studies using Pdr5-enriched plasma membranes. Similar observations have not yet been reported for any other MDR efflux pump. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Through the coupling of nucleotide hydrolysis with drug efflux, Pdr5 homologues enable pathogenic species to survive in the presence of chemically diverse antifungal agents. Our structural and functional results reveal details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across interdomain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens avenues for the development of effective antifungal compounds.

ABCB4 is described as an ATP-binding cassette (ABC) transporter that primarily transports lipids of the phosphatidylcholine (PC) family but is also capable of translocating a subset of typical multidrug-resistance-associated drugs. The high degree of amino acid identity of 76% for ABCB4 and ABCB1, which is a prototype multidrug-resistance-mediating protein, results in ABCB4's second subset of substrates, which overlap with ABCB1's substrates. This often leads to incomplete annotations of ABCB4, in which it was described as exclusively PC-lipid specific. When the hydrophilic amino acids from ABCB4 are changed to the analogous but hydrophobic ones from ABCB1, the stimulation of ATPase activity by 1,2-dioleoyl-sn-glycero-3-phosphocholine, as a prime example of PC lipids, is strongly diminished, whereas the modulation capability of ABCB1 substrates remains unchanged. This indicates two distinct and autonomous substrate binding sites in ABCB4.

The liver is beyond any doubt the most important metabolic organ of the human body. This function requires an intensive crosstalk within liver cellular structures, but also with other organs. Membrane transport proteins are therefore of upmost importance as they represent the sensors and mediators that shuttle signals from outside to the inside of liver cells and/or vice versa. In this review, we summarize the known literature of liver transport proteins with a clear emphasis on functional and structural information on ATP binding cassette (ABC) transporters, which are expressed in the human liver. These primary active membrane transporters form one of the largest families of membrane proteins. In the liver, they play an essential role in for example bile formation or xenobiotic export. Our review provides a state of the art and comprehensive summary of the current knowledge of hepatobiliary ABC transporters. Clearly, our knowledge has improved with a breath-taking speed over the last few years and will expand further. Thus, this review will provide the status quo and will lay the foundation for new and exciting avenues in liver membrane transporter research.

Members of the ATP-binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP-binding cassette in the nucleotide-binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution, the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that currently comprises seven different types based on structural homology in the TMDs.

Summary The ABC-transporters OpuB and OpuC from Bacillus subtilis function as osmoprotectant import systems. Their structural genes have most likely evolved through a duplication event but the two transporters are remarkably different in their substrate profile. OpuB possesses narrow substrate specificity, while OpuC is promiscuous. We assessed the functionality of hybrids between these two ABC-transporters by reciprocally exchanging the coding regions for the OpuBC and OpuCC substrate-binding proteins between the corresponding opuB and opuC operons. Substantiating the critical role of the binding protein in setting the substrate specificity of ABC transporters, OpuB::OpuCC turned into a promiscuous system, while OpuC::OpuBC now exhibited narrow substrate specificity. Both hybrid transporters possessed a high affinity for their substrates but the transport capacity of the OpuB::OpuCC system was moderate due to the synthesis of only low amounts of the xenogenetic OpuCC protein. Suppressor mutations causing single amino acid substitutions in the GbsR repressor controlling the choline to glycine betaine biosynthesis pathway greatly improved OpuB::OpuCC-mediated compatible solute import through transcriptional up-regulation of the hybrid opuB::opuCC operon. Collectively, we demonstrate for the first time that one can synthetically switch the substrate specificity of a given ABC transporter by combining its core components with a xenogenetic ligand-binding protein. This article is protected by copyright. All rights reserved.

Lanthipeptides are ribosomally synthesized and post-translationally modified peptides containing dehydrated amino acids and (methyl-)lanthionine rings. One of the best-studied example is nisin, which is synthesized as a precursor peptide comprising of an N-terminal leader peptide and a C-terminal core peptide. Amongst others, the leader peptide is crucial for enzyme recognition and acts as a secretion signal for the ABC transporter NisT which secrets nisin in a proposed channeling mechanism. Here, we present anin vivosecretion analysis of this process in the presence and absence of the maturation machinery composed of the dehydratase NisB and the cyclase NisC. The data clearly demonstrated that the function of NisC, but the mere presence of NisB modulated the apparent secretion rates. Additional in vitro studies of detergent-solubilized NisT revealed how the activity of this ABC transporter is again influenced by the enzymes of the maturation machinery, but not the substrate.

Resistance to antimicrobial and chemotherapeutic agents is a significant clinical problem. Overexpression of multidrug efflux pumps often creates broad-spectrum resistance in cancers and pathogens. We describe a mutation, A666G, in the yeast ABC transporter Pdr5 that shows greater resistance to most of the tested compounds than does an isogenic wild-type strain. This mutant exhibited enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild type and mutant showed a strong dependence on the ATP concentration, but were about twice as high in the latter. Plots of the initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was further enhanced in the A666G mutant. Resistance to imazalil sulfate was about 3-4x as great in the A666G mutant strain as in the wild type. When this transport substrate was used to inhibit the rhodamine 6G transport, the A666G mutant inhibition curves also showed greater cooperativity than the wild-type strain. Our results suggest a novel and important mechanism: under selection, Pdr5 mutants can increase drug resistance by improving cooperative interactions between drug transport sites.

Multidrug resistance (MDR) in tumors and pathogens remains a major problem in the efficacious treatment of patients by reduction of therapy options and subsequent treatment failure. Various mechanisms are described to be involved in the development of MDR with overexpression of ATP-binding cassette (ABC) transporters reflecting the most extensively studied. These membrane transporters translocate a wide variety of substrates utilizing energy from ATP hydrolysis leading to decreased intracellular drug accumulation and impaired drug efficacy. One treatment strategy might be inhibition of transporter-mediated efflux by small molecules. Isocoumarins and 3,4-dihydroisocoumarins are a large group of natural products derived from various sources with great structural and functional variety, but have so far not been in the focus as potential MDR reversing agents. Thus, three natural products and nine novel 3,4-dihydroisocoumarins were designed and analyzed regarding cytotoxicity induction and inhibition of human ABC transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) in a variety of human cancer cell lines as well as the yeast ABC transporter Pdr5 in Saccharomyces cerevisiae. Dual inhibitors of P-gp and BCRP and inhibitors of Pdr5 were identified, and distinct structure-activity relationships for transporter inhibition were revealed. The strongest inhibitor of P-gp and BCRP, which inhibited the transporters up to 80 to 90% compared to the respective positive controls, demonstrated the ability to reverse chemotherapy resistance in resistant cancer cell lines up to 5.6-fold. In the case of Pdr5, inhibitors were identified that prevented substrate transport and/or ATPase activity with IC50 values in the low micromolar range. However, cell toxicity was not observed. Molecular docking of the test compounds to P-gp revealed that differences in inhibition capacity were based on different binding affinities to the transporter. Thus, these small molecules provide novel lead structures for further optimization.

Background The overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored. Results In E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF and AcrB), which are also recalcitrant contaminants crystallizing even at low concentration. Engineered expression hosts presented an enhanced fitness and improved folding of target membrane proteins, which correlated with an altered membrane fluidity. We demonstrated the scope of this approach by overproducing several membrane proteins (4 different ABC transporters, YidC and SecYEG). Conclusions In summary, E. coli membrane engineering unprecedentedly increases the quality and yield of membrane protein preparations. This strategy opens a new field for membrane protein production, complementary to gene expression tuning. Electronic supplementary material The online version of this article (10.1186/s12934-019-1182-1) contains supplementary material, which is available to authorized users.

Phytohormones play a major role in plant growth and development. They are in most cases not synthesized in their target location and hence need to be transported to the site of action, by for instance ATP-binding cassette transporters. Within the ATP-binding cassette transporter family, Pleiotropic Drug Resistance transporters are known to be involved in phytohormone transport. Interestingly, PDRs are only present in plants and fungi. In contrast to fungi, there are few biochemical studies of plant PDRs and one major reason is that suitable overexpression systems have not been identified. In this study, we evaluate the expression system Pichia pastoris for heterologous overexpression of PDR genes of the model plant Arabidopsis thaliana. We successfully cloned and expressed the potential phytohormone transporters PDR2 and PDR8 in P. pastoris. Sucrose gradient centrifugation confirmed that the overexpressed proteins were correctly targeted to the plasma membrane of P. pastoris and initial functional studies demonstrated ATPase activity for WBC1. However, difficulties in cloning and heterologous overexpression might be particular obstacles of the PDR family, since cloning and overexpression of White Brown Complex 1, a half-size transporter of the same ABCG subfamily with comparable domain organization, was more easily achieved. We present strategies and highlight critical factors to successfully clone plant PDR genes and heterologously expressed in P. pastoris.

ABC (ATP-binding cassette) transporters are ubiquitous integral membrane proteins catalyzing the active export or import of structurally and functionally unrelated compounds. In humans, these proteins are clinically and economically important, as their dysfunction is responsible for a number of diseases. In the case of multidrug resistance (MDR) ABC exporters, they particularly confer resistance to a broad spectrum of toxic compounds, placing them in the focus of clinical research. However, ABC-mediated drug resistance is not only restricted to humans. In yeast for example, MDR is called pleiotropic drug resistance (PDR). Important and well-studied members of the PDR subfamily of ABC transporters are Pdr5 from Saccharomyces cerevisiae and its homolog Cdr1 from Candida albicans. Mutational studies of these two transporters provided many insights into the complexity and conceivable mechanism of the interdomain cross-talk that transmits the energy gained from ATP hydrolysis to the substrate translocation process across the membrane. In this review, we summarize and discuss our current knowledge of the interdomain cross-talk as well as new results obtained for asymmetric ABC transporters and derive possible structural and functional implications for Pdr5.

Type I secretion systems are widespread in Gram-negative bacteria and mediate the one-step translocation of a large variety of proteins serving for diverse purposes, including nutrient acquisition or bacterial virulence. Common to most substrates of type I secretion systems is the presence of a C-terminal secretion sequence that is not cleaved during or after translocation. Furthermore, these protein secretion nanomachineries are always composed of an ABC transporter, a membrane fusion protein, both located in the inner bacterial membrane, and a protein of the outer membrane. These three membrane proteins transiently form a 'tunnel channel' across the periplasmic space in the presence of the substrate. Here we summarize the recent findings with respect to structure, function and application of type I secretion systems.

Overexpression and purification of membrane proteins has been a bottleneck for their functional and structural study for a long time. Both homologous and heterologous expression of membrane proteins with suitable tags for purification presents unique challenges for cloning and expression. Saccharomyces cerevisiae is a potential host system with significant closeness to higher eukaryotes and provides opportunity for attempts to express membrane proteins. In the past, bakers yeast containing mutations within the transcriptional regulator Pdr1 has been used to overexpress various membrane proteins including for example the ABC transporters Pdr5 and Yor1, respectively. In this study we exploited this system and tried to express and purify 3 membrane proteins in yeast along with Pdr5 and Yor1 viz. Rsb1, Mdl1 and Drs2 by virtue of an N-terminal 14-histidine affinity tag. Out of these five, we could express all membrane proteins although at different levels. Satisfactory yields were obtained for three examples i.e. Pdr5, Yor1 and Drs2. Rsb1 expression was comparatively low and Mdl1 was rather unsatisfactory. Thus, we demonstrate here the application of this yeast based expression system that is suitable for cloning, expression and purification of a wide variety of membrane proteins.

Pdr5 is a plasma membrane-bound ABC transporter from Saccharomyces cerevisiae and is involved in the phenomenon of resistance against xenobiotics, which are clinically relevant in bacteria, fungi, and humans. Many fungal ABC transporters such as Pdr5 display an inherent asymmetry in their nucleotide-binding sites (NBS) unlike most of their human counterparts. This degeneracy of the NBSs is very intriguing and needs explanation in terms of structural and functional relevance. In this study, we mutated nonconsensus amino acid residues in the NBSs to its consensus counterpart and studied its effect on the function of the protein and effect on yeast cells. The completely "regenerated" Pdr5 protein was severely impaired in its function of ATP hydrolysis and of rhodamine 6G transport. Moreover, we observe alternative compensatory mechanisms to counteract drug toxicity in some of the mutants. In essence, we describe here the first attempts to restore complete symmetry in an asymmetric ABC transporter and to study its effects, which might be relevant to the entire class of asymmetric ABC transporters.

Type 1 secretion systems (T1SS) of Gram-negative bacteria secrete a broad range of substrates into the extracellular space. Common to all substrates is a C-terminal secretion sequence and nonapeptide repeats in the C-terminal part that bind Ca2+ in the extracellular space, to trigger protein folding. Like all T1SS, the hemolysin A (HlyA) T1SS of Escherichia coli consists of an ABC transporter, a membrane fusion protein and an outer membrane protein allowing the one step translocation of the substrate across both membranes. Here, we analyzed the secretion rate of the HlyA T1SS. Our results demonstrate that the rate is independent of substrate-size and operates at a speed of approximately 16 amino acids per transporter per second. We also demonstrate that the rate is independent of the extracellular Ca2+ concentration raising the question of the driving force of substrate secretion by T1SS in general.

Type 1 secretion systems (T1SS) transport a wide range of substrates across both membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC (ATP-binding cassette) transporter. The ABC transporter HlyB (haemolysin B) is part of a T1SS catalysing the export of the toxin HlyA in E. coli. HlyB consists of the canonical transmembrane and nucleotide-binding domains. Additionally, HlyB contains an N-terminal CLD (C39-peptidase-like domain) that interacts with the transport substrate, but its functional relevance is still not precisely defined. In the present paper, we describe the purification and biochemical characterization of detergent-solubilized HlyB in the presence of its transport substrate. Our results exhibit a positive co-operativity in ATP hydrolysis. We characterized further the influence of the CLD on kinetic parameters by using an HlyB variant lacking the CLD (HlyB∆CLD). The biochemical parameters of HlyB∆CLD revealed an increased basal maximum velocity but no change in substrate-binding affinity in comparison with full-length HlyB. We also assigned a distinct interaction of the CLD and a transport substrate (HlyA1), leading to an inhibition of HlyB hydrolytic activity at low HlyA1 concentrations. At higher HlyA1 concentrations, we observed a stimulation of the hydrolytic activities of both HlyB and HlyB∆CLD, which was completely independent of the interaction of HlyA1 with the CLD. Notably, all observed effects on ATPase activity, which were also analysed in detail by mass spectrometry, were independent of the HlyA1 secretion signal. These results assign an interdomain regulatory role for the CLD modulating the hydrolytic activity of HlyB.

A very large type I polypeptide begins to reel out from a ribosome; minutes later, the still unidentifiable polypeptide, largely lacking secondary structure, is now in some cases a thousand or more residues longer. Synthesis of the final hundred C-terminal residues commences. This includes the identity code, the secretion signal within the last 50 amino acids, designed to dock with a waiting ATP binding cassette (ABC) transporter. What happens next is the subject of this review, with the main, but not the only focus on hemolysin HlyA, an RTX protein toxin secreted by the type I system. Transport substrates range from small peptides to giant proteins produced by many pathogens. These molecules, without detectable cellular chaperones, overcome enormous barriers, crossing two membranes before final folding on the cell surface, involving a unique autocatalytic process. Unfolded HlyA is extruded posttranslationally, C-terminal first. The transenvelope “tunnel” is formed by HlyB (ABC transporter), HlyD (membrane fusion protein) straddling the inner membrane and periplasm and TolC (outer membrane). We present a new evaluation of the C-terminal secretion code, and the structure function of HlyD and HlyB at the heart of this nanomachine. Surprisingly, key details of the secretion mechanism are remarkably variable in the many type I secretion system subtypes. These include alternative folding processes, an apparently distinctive secretion code for each type I subfamily, and alternative forms of the ABC transporter; most remarkably, the ABC protein probably transports peptides or polypeptides by quite different mechanisms. Finally, we suggest a putative structure for the Hly-translocon, HlyB, the multijointed HlyD, and the TolC exit.

The inhibition of ABC (ATP binding cassette) transporters is considered a powerful tool to reverse multidrug resistance. Zosuquidar featuring a difluorocyclopropyl-annulated dibenzosuberyl moiety has been found to be an inhibitor of the P-glycoprotein, one of the best-studied multidrug efflux pumps. Twelve 5-oxyisoquinoline derivatives, which are analogues of zosuquidar wherein the dibenzosuberyl-piperazine moiety is replaced by either a diarylaminopiperidine or a piperidone-derived acetal or thioacetal group, have been synthesized as pure enantiomers. Their inhibitory power has been evaluated for the bacterial multidrug-resistance ABC transporter LmrCD and fungal Pdr5. Four of the newly synthesized compounds reduced the transport activity to a higher degree than zosuquidar, being up to fourfold more efficient than the lead compound in the case of LmrCD and about two times better for Pdr5.

Summary Type 1 secretion systems (T1SS) catalyze the one step protein transport across the membranes of Gram-negative bacteria and are composed of an outer membrane protein, a membrane fusion protein and an ABC transporter. The ABC transporter consists of the canonical nucleotide binding and transmembrane domains. For the toxin hemolysin A (HlyA), the ABC transporter HlyB carries an additional, N-terminal domain sharing about 40% homology to C39 peptidases, but this "C39-like domain" (CLD) is suggested to feature another, yet unknown function. Our functional and structural analysis demonstrates that the CLD is essential for secretion and that it specifically interacts with the unfolded state of HlyA. We determined the nuclear magnetic resonance structure of the CLD as well as the substrate-binding region within the CLD. This mode of action, represents a mechanism within T1SS and answers the question, how a large and unfolded substrate is protected inside the cells during secretion.

Bacillus subtilis can synthesize the compatible solute glycine betaine as an osmoprotectant from an exogenous supply of the precursor choline. Import of choline is mediated by two osmotically inducible ABC transport systems: OpuB and OpuC. OpuC catalyzes the import of various osmoprotectants, whereas OpuB is highly specific for choline. OpuBC is the substrate-binding protein of the OpuB transporter, and we have analyzed the affinity of the OpuBC/choline complex by intrinsic tryptophan fluorescence and determined a K(d) value of about 30 μM. The X-ray crystal structure of the OpuBC/choline complex was solved at a resolution of 1.6 Å and revealed a fold typical of class II substrate-binding proteins. The positively charged trimethylammonium head group of choline is wedged into an aromatic cage formed by four tyrosine residues and is bound via cation-pi interactions. The hydroxyl group of choline protrudes out of this aromatic cage and makes a single interaction with residue Gln19. The substitution of this residue by Ala decreases choline binding affinity by approximately 15-fold. A water network stabilizes choline within its substrate-binding site and promotes indirect interactions between the two lobes of the OpuBC protein. Disruption of this intricate water network by site-directed mutagenesis of selected residues in OpuBC either strongly reduces choline binding affinity (between 18-fold and 25-fold) or abrogates ligand binding. The crystal structure of the OpuBC/choline complex provides a rational for the observed choline specificity of the OpuB ABC importer in vivo and explains its inability to catalyze the import of glycine betaine into osmotically stressed B. subtilis cells.

International audience; Secretion of the Escherichia coli toxin hemolysin A (HlyA) is catalyzed by the membrane protein complex HlyB-HlyD-TolC and requires a secretion sequence located within the last 60 amino acids of HlyA. The Hly translocator complex exports a variety of passenger proteins when fused N-terminal to this secretion sequence. However, not all fusions are secreted efficiently. Here, we demonstrate that the maltose binding protein (MalE) lacking its natural export signal and fused to the HlyA secretion signal is poorly secreted by the Hly system. We anticipated that folding kinetics might be limiting secretion, and we therefore introduced the "folding" mutation Y283D. Indeed this mutant fusion protein was secreted at a much higher level. This level was further enhanced by the introduction of a second MalE folding mutation (V8G or A276G). Secretion did not require the molecular chaperone SecB. Folding analysis revealed that all mutations reduced the refolding rate of the substrate, whereas the unfolding rate was unaffected. Thus, the efficiency of secretion by the Hly system is dictated by the folding rate of the substrate. Moreover, we demonstrate that fusion proteins defective in export can be engineered for secretion while still retaining function.

Substrate-binding proteins (SBP) are associated with a wide variety of protein complexes. The proteins are part of ATP-binding cassette transporters for substrate uptake, ion gradient driven transporters, DNA-binding proteins, as well as channels and receptors from both pro-and eukaryotes. A wealth of structural and functional data is available on SBPs, with over 120 unique entries in the Protein Data Bank (PDB). Over a decade ago these proteins were divided into three structural classes, but based on the currently available wealth of structural data, we propose a new classification into six clusters, based on features of their three-dimensional structure. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Multidrug resistance is a major challenge in the therapy of cancer and pathogenic fungal infections. More than three decades ago, P-glycoprotein was the first identified multidrug transporter. It has been studied extensively at the genetic and biochemical levels ever since. Pdr5, the most abundant ATP-binding cassette transporter in Saccharomyces cerevisiae, is highly homologous to azole-resistance-mediating multidrug transporters in fungal pathogens, and a focus of clinical drug resistance research. Despite functional equivalences, P-glycoprotein and Pdr5 exhibit striking differences in their architecture and mechanisms. In this minireview, we discuss the mechanisms of substrate selection and multidrug transport by comparing the fraternal twins P-glycoprotein and Pdr5. We propose that substrate selection in eukaryotic multidrug ATP-binding cassette transporters is not solely determined by structural features of the transmembrane domains but also by their dynamic behavior.

The periplasmic ligand-binding protein ChoX is part of the ABC transport system ChoVWX that imports choline as a nutrient into the soil bacterium Sinorhizobium meliloti. We have recently reported the crystal structures of ChoX in complex with its ligands choline and acetylcholine and the structure of a fully closed but substrate-free state of ChoX. This latter structure revealed an architecture of the ligand-binding site that is superimposable to the closed, ligand-bound form of ChoX. We report here the crystal structure of ChoX in an unusual, ligand-free conformation that represents a semi-closed form of ChoX. The analysis revealed a subdomain movement in the N-lobe of ChoX. Comparison with the two well-characterized substrate binding proteins, MBP and HisJ, suggests the presence of a similar subdomain in these proteins.

Substrate-binding proteins or extracellular solute receptors (ESRs) are components of both ABC (ATP binding cassette) and TRAP-T (tripartite ATP-independent periplasmic transporter). The TRAP-T system UehABC from Silicibacter pomeroyi DSS-3 imports the compatible solutes ectoine and 5-hydroxyectoine as nutrients. UehA, the ESR of the UehABC operon, binds both ectoine and 5-hydroxyectoine with high affinity (K(d) values of 1.4+/-0.1 and 1.1+/-0.1 microM, respectively) and delivers them to the TRAP-T complex. The crystal structure of UehA in complex with ectoine was determined at 2.9-A resolution and revealed an overall fold common for all ESR proteins from TRAP systems determined so far. A comparison of the recently described structure of TeaA from Halomonas elongata and an ectoine-binding protein (EhuB) from an ABC transporter revealed a conserved ligand binding mode that involves both directed and cation-pi interactions. Furthermore, a comparison with other known TRAP-T ESRs revealed a helix that might act as a selectivity filter imposing restraints on the ESRs that fine-tune ligand recognition and binding and finally might determine the selection of the cognate substrate.

The ABC transporter (ATP-binding-cassette transporter) OpuA is one of five membrane transport systems in Bacillus subtilis that mediate osmoprotection by importing compatible solutes. Just like all bacterial and archaeal ABC transporters that catalyse the import of substrates, OpuA (where Opu is osmoprotectant uptake) is composed of an ATPase subunit (OpuAA), a transmembrane subunit (OpuAB) and an extracellular substrate-binding protein (OpuAC). In contrast with many well-known ABC-ATPases, OpuAA is composed not only of a catalytic and a helical domain but also of an accessory domain located at its C-terminus. The paradigm of such an architecture is MalK, the ABC-ATPase of the maltose importer of Escherichia coli, for which detailed structural and functional information is available. In the present study, we have applied solution FRET (Förster resonance energy transfer) techniques using two single cysteine mutants to obtain initial structural information on the architecture of the OpuAA dimer in solution. Analysing our results in detail and comparing them with the existing MalK structures revealed that the catalytic and helical domains adopted an arrangement similar to those of MalK, whereas profound differences in the three-dimensional orientation of the accessory domain, which contains two CBS (cystathionine β-synthetase) domains, were observed. These results shed new light on the role of this accessory domain present in a certain subset of ABC-ATPase in the fine-tuning of three-dimensional structure and biological function.

The human transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticulum lumen. The functional unit of TAP is a heterodimer composed of the TAP1 and TAP2 subunits, both of which are members of the ABC-transporter family. ABC-transporters are ATP-dependent pumps, channels, or receptors that are composed of four modules: two nucleotide-binding domains (NBDs) and two transmembrane domains (TMDs). Although the TMDs are rather divergent in sequence, the NBDs are conserved with respect to structure and function. Interestingly, the NBD of TAP1 contains mutations at amino acid positions that have been proposed to be essential for catalytic activity. Instead of a glutamate, proposed to act as a general base, TAP1 contains an aspartate and a glutamine instead of the conserved histidine, which has been suggested to act as the linchpin. We used this degeneration to evaluate the individual contribution of these two amino acids to the ATPase activity of the engineered TAP1-NBD mutants. Based on our results a catalytic hierarchy of these two fundamental amino acids in ATP hydrolysis of the mutated TAP1 motor domain was deduced.

The ATP-binding cassette (ABC)-transporter haemolysin (Hly)B, a central element of a Type I secretion machinery, acts in concert with two additional proteins in Escherichia coli to translocate the toxin HlyA directly from the cytoplasm to the exterior. The basic set of crystal structures necessary to describe the catalytic cycle of the isolated HlyB-NBD (nucleotide-binding domain) has now been completed. This allowed a detailed analysis with respect to hinge regions, functionally important key residues and potential energy storage devices that revealed many novel features. These include a structural asymmetry within the ATP dimer that was significantly enhanced in the presence of Mg2+, indicating a possible functional asymmetry in the form of one open and one closed phosphate exit tunnel. Guided by the structural analysis, we identified two amino acids, closing one tunnel by an apparent salt bridge. Mutation of these residues abolished ATP-dependent cooperativity of the NBDs. The implications of these new findings for the coupling of ATP binding and hydrolysis to functional activity are discussed.

The ATPase activity of the ABC (ATP-binding cassette) ATPase domain of the HlyB (haemolysin B) transporter is required for secretion of Escherichia coli haemolysin via the type I pathway. Although ABC transporters are generally presumed to function as dimers, the precise role of dimerization remains unclear. In the present study, we have analysed the HlyB ABC domain, purified separately from the membrane domain, with respect to its activity and capacity to form physically detectable dimers. The ATPase activity of the isolated ABC domain clearly demonstrated positive co-operativity, with a Hill coefficient of 1.7. Furthermore, the activity is (reversibly) inhibited by salt concentrations in the physiological range accompanied by proportionately decreased binding of 8-azido-ATP. Inhibition of activity with increasing salt concentration resulted in a change in flexibility as detected by intrinsic tryptophan fluorescence. Finally, ATPase activity was sensitive towards orthovanadate, with an IC50 of 16 μM, consistent with the presence of transient dimers during ATP hydrolysis. Nevertheless, over a wide range of protein or of NaCl or KCl concentrations, the ABC ATPase was only detected as a monomer, as measured by ultracentrifugation or gel filtration. In contrast, in the absence of salt, the sedimentation velocity determined by analytical ultracentrifugation suggested a rapid equilibrium between monomers and dimers. Small amounts of dimers, but apparently only when stabilized by 8-azido-ATP, were also detected by gel filtration, even in the presence of salt. These data are consistent with the fact that monomers can interact at least transiently and are the important species during ATP hydrolysis.

Adaptation of microorganisms to changing osmotic conditions is a prerequisite for survival and cellular vitality for most microorganisms. In the Gram-positive soil bacterium Bacillus subtilis, five transport systems catalyze the uptake of compatible solutes across the plasma membrane that allow the growth of B. subtilis over a wide range of osmotic conditions. Focus of this review is the osmoprotectant uptake A (OpuA) transporter, a member of the family of substrate-binding protein (SBP)-dependent ATP-binding cassette (ABC) transporters that mediates the uptake of the compatible solutes glycine betaine and proline betaine. OpuA is composed of three subunits: a nucleotide-binding domain (OpuAA) located in the cytosol, a transmembrane domain (OpuAB), and a SBP (OpuAC), which binds glycine betaine and proline betaine with high specificity and targets it to OpuAB for ATP-dependent translocation across the plasma membrane. After a brief introduction in the field of bacterial osmoadaptation, we will summarize our recent findings about the biochemical and structural analysis of the components of the OpuA systems. Our studies covered both the isolated subunits of the OpuA transporter and initial investigations of the whole transporter in vitro.

The ABC-transporter haemolysin B is a central component of the secretion machinery that translocates the toxin, haemolysin A, in a Sec-independent fashion across both membranes of E. coli . Here, we report the X-ray crystal structure of the nucleotide-binding domain (NBD) of HlyB. The molecule shares the common overall architecture of ABC-transporter NBDs. However, the last three residues of the Walker A motif adopt a 3 10 helical conformation, stabilized by a bound anion. In consequence, this results in an unusual interaction between the Walker A lysine residue and the Walker B glutamate residue. As these residues are normally required to be available for ATP binding, for catalysis and for dimer formation of ABC domains, we suggest that this conformation may represent a latent monomeric form of the NBD. Surprisingly, comparison of available NBD structures revealed a structurally diverse region (SDR) of about 30 residues within the helical arm II domain, unique to each of the eight NBDs analyzed. As this region interacts with the transmembrane part of ABC-transporters, the SDR helps to explain the selectivity and/or targeting of different NBDs to their cognate transmembrane domains.

A member of the family of RTX toxins, Escherichia coli haemolysin A, is secreted from Gram-negative bacteria. It carries a C-terminal secretion signal of approximately 50 residues, targeting the protein to the secretion or translocation complex, in which the ABC-transporter HlyB is a central element. We have purified the nucleotide-binding domain of HlyB (HlyB-NBD) and a C-terminal 23kDa fragment of HlyA plus the His-tag (HlyA1), which contains the secretion sequence. Employing surface plasmon resonance, we were able to demonstrate that the HlyB-NBD and HlyA1 interact with a K(D) of approximately 4 microM. No interaction was detected between the HlyA fragment and unrelated NBDs, OpuAA, involved in import of osmoprotectants, and human TAP1-NBD, involved in the export of antigenic peptides. Moreover, a truncated version of HlyA1, lacking the secretion signal, failed to interact with the HlyB-NBD. In addition, we showed that ATP accelerated the dissociation of the HlyB-NBD/HlyA1 complex. Taking these results together, we propose a model for an early stage of initiation of secretion in vivo, in which the NBD of HlyB, specifically recognizes the C terminus of the transport substrate, HlyA, and where secretion is initiated by subsequent displacement of HlyA from HlyB by ATP.

ATP-binding cassette (ABC) transporters are central to many physiological processes, including the uptake of nutrients, the non-classical secretion of signaling molecules and toxins, multidrug resistance and the development of human disease. As one might expect from this spectrum of translocation events, these ubiquitous, ATP-dependent pumps or channels are capable of transporting an enormous variety of substrates, ranging from small ions to large proteins. Recently determined structures of full-length ABC transporters and isolated ABC domains have increased our understanding of the functional mechanism of these proteins.

ATP-binding cassette (ABC) transporters are ubiquitous integral membrane proteins, which catalyze the translocation of molecules across biological membranes in an ATP-dependent manner. Despite the diversity in the transported substrates, they all share the same architecture, comprised of two transmembrane (TMD) and two nucleotide-binding domains (NBD). Members of the bacteriocin ABC transporter subfamily feature a special domain, belonging to the C39 (cystein protease family 39) peptidase protein family. These domains are assumed to cleave a C-terminal signal sequence from the protein or peptide substrate before or during the transport process. Although the C39 peptidase-like domain of the ABC transporter haemolysin B from E. coli shows no proteolytic activity, it is essential for the function of this transporter. In order to elucidate the contribution of the isolated C39 peptidase-like domain in the whole transport process, the backbone and side chain (1)H, (15)N and (13)C-NMR chemical shifts have been assigned.

The human multidrug resistance protein 3 (MDR3/ABCB4) belongs to the ubiquitous family of ATP-binding cassette (ABC) transporters and is located in the canalicular membrane of hepatocytes. There it flops the phospholipids of the phosphatidylcholine (PC) family from the inner to the outer leaflet. Here, we report the characterization of wild type MDR3 and the Q1174E mutant, which was identified previously in a patient with progressive familial intrahepatic cholestasis type 3 (PFIC-3). We expressed different variants of MDR3 in the yeast Pichia pastoris, purified the proteins via tandem affinity chromatography, and determined MDR3-specific ATPase activity in the presence or absence of phospholipids. The ATPase activity of wild type MDR3 was stimulated 2-fold by liver PC or 1,2-dioleoyl-sn-glycero-3-phosphatidylethanolamine lipids. Furthermore, the cross-linking of MDR3 with a thiol-reactive fluorophore blocked ATP hydrolysis and exhibited no PC stimulation. Similarly, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin lipids did not induce an increase of wild type MDR3 ATPase activity. The phosphate analogues beryllium fluoride and aluminum fluoride led to complete inhibition of ATPase activity, whereas orthovanadate inhibited exclusively the PC-stimulated ATPase activity of MDR3. The Q1174E mutation is located in the nucleotide-binding domain in direct proximity of the leucine of the ABC signature motif and extended the X loop, which is found in ABC exporters. Our data on the Q1174E mutant demonstrated basal ATPase activity, but PC lipids were incapable of stimulating ATPase activity highlighting the role of the extended X loop in the cross-talk of the nucleotide-binding domain and the transmembrane domain.

The hepatocyte contains a battery of membrane transporters in the sinusoidal and canalicular membrane that guarantee the regulated import and export of nutrients, messengers and toxic compounds. In the canalicular membrane, a number of important ABC (ATP binding cassette) transporters are located. Three of these primary active transporters, BSEP (bile salt export pump, ABC B11), MDR3 (multidrug resistance protein 3, ABC B4) and ABC G5/G8 catalyze the secretion of bile acids (BSEP), phosphatidylcholine (MDR3) and cholesterol (ABC G5/G8). These three molecules form mixed micelles in bile and thereby reduce the harmful action of unbound bile salts on membranes. BSEP, which was initially named s-Pgp (sister of P-gp) and MDR3 share a high sequence identity with MDR1 (P-glycoprotein, ABC B1), which is the hallmark of the family of multidrug resistance ABC transporter present in every eukaryotic cell. For BSEP the identity is approximately 70% and for MDR3 even 85%, respectively. Nevertheless, MDR3 is specific for phosphatidylcholine lipids and BSEP is specific for bile acids, while MDR1 is capable of translocating a myriad of structurally unrelated compound across biological membranes. This of course raises the question, which residues impose specificity in the case of BSEP and MDR3 and which residues impose promiscuity in the case of MDR1. Homology models of BSEP and MDR3 were generated based on the crystal structures of Sav1866 and mouse P-gp to obtain initial information about the putative three-dimensional location of certain amino acid residues of BSEP and MDR3. However, one has to stress that these “structures” represent only models that have to be used with proper caution. The homology model of MDR3 was helpful in providing a structural explanation for the non-functionality of the MDR3 mutant H1231Y. The mutant protein was properly located to the plasma membrane, but displayed a severe substrate transport phenotype. Histidine 1231 corresponds to the histidine of the H-loop, a residue that is critical for ATP hydrolysis in many other ABC transporter systems. Thus, exchange of His against Tyr in position 1231 of MDR3 will impair ATP hydrolysis in one of the two ATP binding site drastically reducing transport activity of MDR3 H1231Y [1]. To study the transport cycle and the importance of mutations within these transporters on their function, overexpression systems are required. We decided to use the methylotrophic yeast Pichia pastoris for the overexpression of two of the three transporters, BSEP and MDR3, respectively. Initially we encountered severe problems in cloning the transporters genes in E. coli employing standard approaches. Only after establishing a cloning strategy that is entirely based on Saccharomyces cerevisiae, both genes could be successfully cloned [2]. This allowed us to combine the speed and ease of cloning of this yeast system for the wild type genes and mutations that were identified in patients. Having this tool in hand, we overexpressed BSEP in yeast to study its transport function in plasma membrane vesicles. This allowed us to establish structure-function relations for a new clinically relevant mutation (G374S) that resulted in a phenotype between PFIC-2 and BRIC-2. Although membrane targeting was normal, e.g. targeting to the canalicular membrane was evident, transport activity of the G374S was strongly impaired [3]. The Sav1866-based homology model of BSEP suggested that Gly 374 is lining the translocation pore of BSEP. Thus, an exchange to serine would impose steric restriction within the pore, thereby influencing the substrate spectrum as evident from the bile acid transport assays. Based on the expression levels of BSEP and MDR3, purification protocols for both membrane proteins were successfully established [4]. Thus, next to the transport assay that is based only highly enriched yeast plasma membranes, purified protein is now also available in the detergent-solubilized state. While the transport assay can be used for BSEP to analyze a possible influence of a mutation on the transporter activity of the protein, purified MDR3 can be used to determine the influence of a mutation on ATPase activity and / or stimulation or inhibition of this activity. In summary, the established yeast overexpression system combines the ease and speed of yeast cloning approaches with the possibility to overexpress and purify both proteins in a quantity and homogeneity sufficient for biochemical and biophysical investigations of the function of these two ABC transporters. Furthermore, the speed with which mutants can be obtained will allow an in depth characterization of clinically relevant mutations of both transporters with respect to function and spectrum of transported substrates.

The human liver ATP-binding cassette (ABC) transporters bile salt export pump (BSEP/ABCB11) and the multidrug resistance protein 3 (MDR3/ABCB4) fulfill the translocation of bile salts and phosphatidylcholine across the apical membrane of hepatocytes. In concert with ABCG5/G8, these two transporters are responsible for the formation of bile and mutations within these transporters can lead to severe hereditary diseases. In this study, we report the heterologous overexpression and purification of human BSEP and MDR3 as well as the expression of the corresponding C-terminal GFP-fusion proteins in the yeast Pichia pastoris. Confocal laser scanning microscopy revealed that BSEP-GFP and MDR3-GFP are localized in the plasma membrane of P. pastoris. Furthermore, we demonstrate the first purification of human BSEP and MDR3 yielding ∼1 mg and ∼6 mg per 100 g of wet cell weight, respectively. By screening over 100 detergents using a dot blot technique, we found that only zwitterionic, lipid-like detergents such as Fos-cholines or Cyclofos were able to extract both transporters in sufficient amounts for subsequent functional analysis. For MDR3, fluorescence-detection size exclusion chromatography (FSEC) screens revealed that increasing the acyl chain length of Fos-Cholines improved monodispersity. BSEP purified in n-dodecyl-β-D-maltoside or Cymal-5 after solubilization with Fos-choline 16 from P. pastoris membranes showed binding to ATP-agarose. Furthermore, detergent-solubilized and purified MDR3 showed a substrate-inducible ATPase activity upon addition of phosphatidylcholine lipids. These results form the basis for further biochemical analysis of human BSEP and MDR3 to elucidate the function of these clinically relevant ABC transporters.

Secretion is an essential task for prokaryotic organisms to interact with their surrounding environment. In particular, the production of extracellular proteins and peptides is important for many aspects of an organism's survival and adaptation to its ecological niche. In Gram-negative bacteria, six different protein secretion systems have been identified so far, named Type I to Type VI; differing greatly in their composition and mechanism of action (Economou et al., 2006). The two membranes present in Gram-negative bacteria are negotiated either by one-step transport mechanisms (Type I and Type III), where the unfolded substrate is translocated directly into the extracellular space, without any periplasmic intermediates, or by two-step mechanisms (Type II and Type V), where the substrate is first transported into the periplasm to allow folding before a second transport step across the outer membrane occurs. Here we focus on Type I secretion systems and summarise our current knowledge of these one-step transport machineries with emphasis on the N-terminal extensions found in many Type I-specific ABC transporters. ABC transporters containing an N-terminal C39 peptidase domain cut off a leader peptide present in the substrate prior to secretion. The function of the second type of appendix, the C39 peptidase-like domain (CLD), is not yet completely understood. Recent results have shown that it is nonetheless essential for secretion and interacts specifically with the substrate of the transporter. The third group present does not contain any appendix. In light of this difference we compare the function of the appendix and the differences that might exist among the three families of T1SS.

Experimental evidence has been provided that a histidine-loop within the nucleotide binding domain of ABC transporter is essential for efficient function of this class of transporter proteins. Here we report the first patient with a mutation of the putative histidine-loop of a human ABC transporter, the multi drug resistance protein 3 (MDR3). The patient presented at the age of 4 years with a history of severe pruritus, elevated serum gamma-glutamyltransferase and bile acid levels since several years suggesting the diagnosis of progressive familial intrahepatic cholestasis type 3 (PFIC-3) due to defects in MDR3. Liver biopsy demonstrated an apparently normal MDR3 expression, however, genetic analysis revealed a novel homozygous mutation in the ABCB4 gene (c.3691C>T) in the patient. This mutation was associated with a change of histidine to tyrosine at amino acid position 1231 of MDR3 (p.H1231Y). As shown by sequence alignment, this amino acid corresponds to the highly conserved histidine of the "H-loop", which is critical for ATP-hydrolysis, suggesting an essential role of histidine 1231 of human MDR3.

Haemolysin B (HlyB) is a transmembrane protein which belongs to the superfamily of ABC transporters. In vivo, it mediates the non-classical translocation of the 107 kDa toxin HlyA across both membranes of Escherichia coli together with haemolysin D and the outer membrane protein TolC. The cytosolic ATP-binding domain of HlyB has been overexpressed and purified as an N-terminal His-tag fusion protein. Here, the crystallization of the ATPase domain of HlyB in the presence of ATP is described. A native data set has been obtained at a resolution of 2.8 A. Crystals belong to the primitive tetragonal space group P4(x)2(1)2, where x is very likely to be 1 or 3, with unit-cell parameters a = b = 104.6, c = 125.8 A, alpha = beta = gamma = 90 degrees.