Prokaryotic ATP-binding Cassette Transporters Research Articles

MsbA: an ABC transporter paradigm.

ATP-binding cassette (ABC) transporters play an important role in various cellular processes. They display a similar architecture and share a mechanism which couples ATP hydrolysis to substrate transport. However, in the light of current data and recent experimental progress, this protein superfamily appears as multifaceted as their broad substrate range. Among the prokaryotic ABC transporters, MsbA can serve as a paradigm for research in this field. It is located in the inner membrane of Gram-negative bacteria and functions as a floppase for the lipopolysaccharide (LPS) precursor core-LPS, which is involved in the biogenesis of the bacterial outer membrane. While MsbA shows high similarity to eukaryotic ABC transporters, its expression in Gram-negative bacteria makes it conveniently accessible for many experimental approaches from spectroscopy to 3D structure determination. As an essential protein for bacterial membrane integrity, MsbA has also become an attractive target for the development of novel antibiotics. Furthermore, it serves as a model for multidrug efflux pumps. Here we provide an overview of recent findings and their relevance to the field, highlight the potential of methods such as solid-state NMR and EPR spectroscopy and provide a perspective for future work.

ABCA4 is an N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine importer

ATP-binding cassette (ABC) transporters comprise a superfamily of proteins, which actively transport a variety of compounds across cell membranes. Mammalian and most eukaryotic ABC transporters function as exporters, flipping or extruding substrates from the cytoplasmic to the extracellular or lumen side of cell membranes. Prokaryotic ABC transporters function either as exporters or importers. Here we show that ABCA4, an ABC transporter found in retinal photoreceptor cells and associated with Stargardt macular degeneration, is a novel importer that actively flips N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic leaflet of disc membranes, thereby facilitating the removal of potentially toxic retinoid compounds from photoreceptors. ABCA4 also actively transports phosphatidylethanolamine in the same direction. Mutations known to cause Stargardt disease decrease N-retinylidene-phosphatidylethanolamine and phosphatidylethanolamine transport activity of ABCA4. These studies provide the first direct evidence for a mammalian ABC transporter that functions as an importer and provide insight into mechanisms underlying substrate transport and the molecular basis of Stargardt disease.

ABC transporters: the power to change

ATP-binding cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane proteins that are responsible for the ATP-powered translocation of many substrates across membranes. The highly conserved ABC domains of ABC transporters provide the nucleotide-dependent engine that drives transport. By contrast, the transmembrane domains that create the translocation pathway are more variable. Recent structural advances with prokaryotic ABC transporters have provided a qualitative molecular framework for deciphering the transport cycle. An important goal is to develop quantitative models that detail the kinetic and molecular mechanisms by which ABC transporters couple the binding and hydrolysis of ATP to substrate translocation.

3-D structural and functional characterization of the purified KATP channel complex Kir6.2-SUR1.

ATP-sensitive potassium (K(ATP)) channels conduct potassium ions across cell membranes and thereby couple cellular energy metabolism to membrane electrical activity. Here, we report the heterologous expression and purification of a functionally active K(ATP) channel complex composed of pore-forming Kir6.2 and regulatory SUR1 subunits, and determination of its structure at 18 A resolution by single-particle electron microscopy. The purified channel shows ATP-ase activity similar to that of ATP-binding cassette proteins related to SUR1, and supports Rb(+) fluxes when reconstituted into liposomes. It has a compact structure, with four SUR1 subunits embracing a central Kir6.2 tetramer in both transmembrane and cytosolic domains. A cleft between adjacent SUR1s provides a route by which ATP may access its binding site on Kir6.2. The nucleotide-binding domains of adjacent SUR1 appear to interact, and form a large docking platform for cytosolic proteins. The structure, in combination with molecular modelling, suggests how SUR1 interacts with Kir6.2.

Three-dimensional Structure of P-glycoprotein: THE TRANSMEMBRANE REGIONS ADOPT AN ASYMMETRIC CONFIGURATION IN THE NUCLEOTIDE-BOUND STATE

Multidrug resistance of cancer cells and pathogens is a serious clinical problem. A major factor contributing to drug resistance in cancer is the over-expression of P-glycoprotein, a plasma membrane ATP-binding cassette (ABC) drug efflux pump. Three-dimensional structural data with a resolution limit of approximately 8 A have been obtained from two-dimensional crystals of P-glycoprotein trapped in the nucleotide-bound state. Each of the two transmembrane domains of P-glycoprotein consists of six long alpha-helical segments. Five of the alpha-helices from each transmembrane domain are related by a pseudo-2-fold symmetry, whereas the sixth breaks the symmetry. The two alpha-helices positioned closest to the (pseudo-) symmetry axis at the center of the molecule appear to be kinked. A large loop of density at the extracellular surface of the transporter is likely to correspond to the glycosylated first extracellular loop, whereas two globular densities at the cytoplasmic side correspond to the hydrophilic, nucleotide-binding domains. This is the first three-dimensional structure for an intact eukaryotic ABC transporter. Comparison with the structures of two prokaryotic ABC transporters suggests significant differences in the packing of the transmembrane alpha-helices within this protein family.

Functional Importance of Polar and Charged Amino Acid Residues in Transmembrane Helix 14 of Multidrug Resistance Protein 1 (MRP1/ABCC1): IDENTIFICATION OF AN ASPARTATE RESIDUE CRITICAL FOR CONVERSION FROM A HIGH TO LOW AFFINITY SUBSTRATE BINDING STATE

Human multidrug resistance protein 1 (MRP1) confers resistance to many chemotherapeutic agents and transports diverse conjugated organic anions. We previously demonstrated that Glu1089 in transmembrane (TM) 14 is critical for the protein to confer anthracycline resistance. We have now assessed the functional importance of all polar and charged amino acids in this TM helix. Asn1100, Ser1097, and Lys1092, which are all predicted to be on the same face of the helix as to Glu1089, are involved in determining the substrate specificity of the protein. Notably, elimination of the positively charged side chain of Lys1092, increased resistance to the cationic drugs vincristine and doxorubicin, but not the electroneutral drug etoposide (VP-16). In addition, mutations S1097A and N1100A selectively decreased transport of 17beta-estradiol 17-(beta-d-glucuronide) (E217betaG) but not cysteinyl leukotriene 4 (LTC4), demonstrating the importance of multiple residues in this helix in determining substrate specificity. In contrast, mutations of Asp1084 that eliminate the carboxylate side chain markedly decreased resistance to all drugs tested, as well as transport of both E217betaG and LTC4, despite the fact that LTC4 binding was unaffected. We show that these mutations prevent the ATP-dependent transition of the protein from a high to low affinity substrate binding state and drastically diminish ADP trapping at nucleotide binding domain 2. Based on results presented here and crystal structures of prokaryotic ATP binding cassette transporters, Asp1084 may be critical for interaction between the cytoplasmic loop connecting TM13 and TM14 and a region of nucleotide binding domain 2 between the conserved Walker A and ABC signature motifs.

The detergent-soluble maltose transporter is activated by maltose binding protein and verapamil.

The maltose transporter FGK2 complex of Escherichia coli was purified with the aid of a glutathione S-transferase molecular tag. In contrast to the membrane-associated form of the complex, which requires liganded maltose binding protein (MBP) for ATPase activity, the purified detergent-soluble complex exhibited a very high level of ATPase activity. This uncoupled activity was not due to dissociation of the MalK ATPase subunit from the integral membrane protein MalF and MalG subunits. The detergent-soluble ATPase activity of the complex could be further stimulated by wild-type MBP but not by a signaling-defective mutant MBP. Wild-type MBP increased the V(max) of the ATPase 2.7-fold but had no effect on the K(m) of the enzyme for ATP. When the detergent-soluble complex was reconstituted in proteoliposomes, it returned to being dependent on MBP for activation of ATPase, consistent with the idea that the structural changes induced in the complex by detergent that result in activation of the ATPase are reversible. The uncoupled ATPase activity resembled the membrane-bound activity of the complex also with respect to sensitivity to NaN(3), as well as a mercurial, p-chloromercuribenzosulfonic acid. Verapamil, a compound that activates the ATPase activity of the multiple drug resistance P-glycoprotein, activated the maltose transporter ATPase as well. The activation of this bacterial transporter by verapamil suggests that a structural feature that is conserved among both eukaryotic and prokaryotic ATP binding cassette transporters is responsible for this activation.

Classification and Analysis of Eukaryotic ABC Transporters in Complete Eukarya Genomes

We performed classification and analysis of the eukaryotic ABC (ATP-Binding Cassette) transporters in S. cerevisiae, C. elegans, and D. melanogaster which are the three eukarya whose genomes were sequenced completely. The ABC transporters form a major class of active transporters which are widespread in archaea, bacteria, and eukarya. These transporters convey a wide variety of substrates, such as peptides, amino acids, simple sugars, sulfate, phosphate and various drugs. The structure of a prokaryotic ABC transporter usually consists of three components. A typical transporter consists of two integral membrane proteins each having six transmembrane segments, two peripheral proteins that bind and hydrolyze ATP, and a periplasmic (or lipoprotein) substrate binding protein. Many of the genes for the three components form operons as in fact observed in known archaea and bacteria genomes On the other hand, in a typical eukaryotic ABC transporter, the membrane spanning protein and the ATP-binding protein are fused, forming a polypeptide with the membrane spanning domain and the ATP-binding domain. In prokaryotic ABC transporters, the ATP-binding protein component is the most conserved, the membrane protein component is somewhat less conserved, and the substrate-binding component is most divergent in terms of the sequence similarity (Tam and Saier 1993; Saurin and Dassa 1994). Therefore, in eukaryotic ABC transporters, it could also be expected that ATP-binding domain is the most conserved domain. In this analysis, we first searched and compared the eukaryotic ABC transporters in three eukarya complete sequenced genomes, S. cerevisiae, C.elegans and D.melangaster. Next, they were classified into orthologs and paralogs from sequence similarity and domain structure according to the hierarchical cluster analysis. Furthermore, hidden Markov models were built using individual clusters, and they were used to search for similar sequences in other genomes in the KEGG/GENES database.

Genetic and biochemical characterization of a high-affinity betaine uptake system (BusA) in Lactococcus lactis reveals a new functional organization within bacterial ABC transporters.

The cytoplasmic accumulation of exogenous betaine stimulates the growth of Lactococcus lactis cultivated under hyperosmotic conditions. We report that L. lactis possesses a single betaine transport system that belongs to the ATP-binding cassette (ABC) superfamily of transporters. Through transposon mutagenesis, a mutant deficient in betaine transport was isolated. We identified two genes, busAA and busAB, grouped in an operon, busA (betaine uptake system). The transcription of busA is strongly regulated by the external osmolality of the medium. The busAA gene codes for the ATP-binding protein. busAB encodes a 573-residue polypeptide which presents two striking features: (i) a fusion between the regions encoding the transmembrane domain (TMD) and the substrate-binding domain (SBD) and (ii) a swapping of the SBD subdomains when compared to the Bacillus subtilis betaine-binding protein, OpuAC. BusA of L. lactis displays a high affinity towards betaine (K(m) = 1.7 microM) and is an osmosensor whose activity is tightly regulated by external osmolality, leading the betaine uptake capacity of L. lactis to be under dual control at the biochemical and genetic levels. A protein presenting the characteristics predicted for BusAB was detected in the membrane fraction of L. lactis. The fusion between the TMD and the SBD is the first example of a new organization within prokaryotic ABC transporters.

Cloning and sequence analysis of a novel member of the ATP-binding cassette (ABC) protein gene family from Plasmodium falciparum

We have employed oligonucleotide primers directed against the Walker A and B ATP-binding consensus motifs in a PCR-approach to clone a novel member of the eukaryotic ABC protein family of genes from Plasmodium falciparum. The novel gene is predicted to encode a 95.5-kDa protein with two ATP-binding folds each containing a Walker A and B consensus motif and an ABC protein signature sequence. The predicted protein is highly hydrophilic and contains numerous phosphorylation consensus sites but does not contain any potential membrane spanning domains. The gene is present on chromosome 11 and is expressed as a 3.3-kb transcript. The closest homologue with known function to the plasmodial gene is the yeast GCN20 gene which is part of the translation initiation pathway in amino acid starved yeast cells. We have therefore tentatively named the gene Plasmodium falciparum GCN20 homologue ( pfgcn20). The pfgcn20 encoded Pfgcn20 protein is also highly homologous to a number of ATP-binding subunits of prokaryotic ABC transporters. We speculate that Pfgcn20 may be an example of a eukaryotic ATP-binding cytosolic subunit of a multipeptide ABC transporter.

Protein exporter function and in vitro ATPase activity are correlated in ABC‐domain mutants of HlyB

The Escherichia coli toxin exporter HlyB comprises an integral membrane domain fused to a cytoplasmic domain of the ATP-binding cassette (ABC) super-family, and it directs translocation of the 110kDa haemolysin protein out of the bacterial cell without using an N-terminal secretion signal peptide. We have exploited the ability to purify the soluble HlyB ABC domain as a fusion with glutathione S-transferase to obtain a direct correlation of the in vivo export of protein by HlyB with the degree of ATP binding and hydrolysis measured in vitro. Mutations in residues that are invariant or highly conserved in the ATP-binding fold and glycine-rich linker peptide of prokaryotic and eukaryotic ABC transporters caused a complete loss of both HlyB exporter function and ATPase activity in proteins still able to bind ATP effectively and undergo ATP-induced conformational change. Mutation of less-conserved residues caused reduced export and ATP hydrolysis, but not ATP binding, whereas substitutions of poorly conserved residues did not impair activity either in vivo or in vitro. The data show that protein export by HlyB has an absolute requirement for the hydrolysis of ATP bound by its cytoplasmic domain and indicate that comparable mutations that disable other prokaryotic and eukaryotic ABC transporters also cause a specific loss of enzymatic activity.

Structure, Function and Regulation of the Nitrate Transport System of the Cyanobacterium Synechococcus sp. PCC7942

The active nitrate transport system of the cyanobacterium Synechococcus sp. PCC7942 is encoded by the four genes nrtA, nrtB, nrtC and nrtD. It is essential for the growth of the cyanobacterium at physiological concentrations of nitrate and has been shown to be involved in the active transport of nitrite as well. The deduced amino acid sequences of the NrtB, NrtC and NrtD proteins indicate that the transporter is a member of the ABC (ATP-binding cassette) superfamily of active transporters. Among the prokaryotic ABC transporters, the cyanobacterial nitrate/nitrite transporter is unique in having a membrane-bound protein NrtA and an NrtA-like extra domain linked to one of the ATP-binding subunits (C-terminal domain of NrtC). Molecular biological, biochemical and physiological studies suggest that NrtA is the substrate-binding protein required for the transport of nitrate/nitrite and that the C-terminal domain of NrtC has a regulatory role. Comparison of the structures of nitrate transporters from eukaryotic and prokaryotic, photosynthetic and non-photosynthetic organisms indicate that the nrt nitrate/nitrite transporter represents a prokaryotic nitrate transporter distinct from the nitrate transporters of eukaryotes.