Building an understanding of cystic fibrosis on the foundation of ABC transporter structures

Cystic fibrosis (CF) is a fatal disease affecting the lungs and digestive system by impairment of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). While over 1000 mutations in CFTR have been associated with CF, the majority of cases are linked to the deletion of phenylalanine 508 (ΔF508). F508 is located in the first nucleotide binding domain (NBD1) of CFTR. This mutation is sufficient to impair the trafficking of CFTR to the plasma membrane and, thus, its function. As an ABC transporter, recent structural data from the family provide a framework on which to consider the effect of the ΔF508 mutation on CFTR. There are fifty-seven known structures of ABC transporters and domains thereof. Only six of these structures are of the intact transporters. In addition, modern bioinformatic tools provide a wealth of sequence and structural information on the family. We will review the structural information from the RCSB structure repository and sequence databases of the ABC transporters. The available structural information was used to construct a model for CFTR based on the ABC transporter homologue, Sav1866, and provide a context for understanding the molecular pathology of Cystic Fibrosis.     

Junker: an intergenic explorer for bacterial genomes

In the past few decades, scientists from all over the world have taken a keen interest in novel functional units such as small regulatory RNAs, small open reading frames, pseudogenes, transposons, integrase binding attB/attP sites, repeat elements within the bacterial intergenic regions (IGRs) and in the analysis of those "junk" regions for genomic complexity. Here we have developed a web server, named Junker, to facilitate the in-depth analysis of IGRs for examining their length distribution, four-quadrant plots, GC percentage and repeat details. Upon selection of a particular bacterial genome, the physical genome map is displayed as a multiple loci with options to view any loci of interest in detail. In addition, an IGR statistics module has been created and implemented in the web server to analyze the length distribution of the IGRs and to understand the disordered grouping of IGRs across the genome by generating the four-quadrant plots. The proposed web server is freely available at the URL http://pranag.physics.iisc.ernet.in/junker/.     

Effects of fluconazole on Candida glabrata biofilms and its relationship with ABC transporter gene expression

Candida glabrata has emerged as the second most prevalent fungal pathogen and its ability to form biofilms has been considered one of the most important virulence factors, since biofilms present a high tolerance to antifungal agents used in fungal infection treatment. The mechanisms of biofilm tolerance to antifungal agents remain poorly understood. Thus, the aim of this study was to evaluate the effects of fluconazole (FLU) on the formation and control of C. glabrata biofilms and its relation with the expression of genes encoding for ABC transporters, CDR1, SNQ2, and PDR1. For that, minimal inhibitory concentration values for seven C. glabrata strains were determined and the effect of FLU against C. glabrata biofilms was evaluated by total biomass quantification and viable cell enumeration. Matrices from biofilms were analyzed in terms of protein, carbohydrate and DNA content. ABC transporter gene expression was analyzed for quantitative real-time PCR. In addition to the high amounts of proteins and carbohydrates detected in the extracellular matrices in the presence of FLU, this work showed that the overexpression of efflux pumps is a possible mechanism of biofilm tolerance to FLU and this phenomenon alters the structure of C. glabrata biofilms by creating cell clusters.     

Availability and applications of <Emphasis Type="Italic">A</Emphasis>TP-binding <Emphasis Type="Italic">c</Emphasis>assette (ABC) transporter blockers

ATP-binding cassette (ABC) transporters encompass membrane transport proteins that couple the energy derived from ATP hydrolysis to the translocation of solutes across biological membranes. The functions of these proteins include ancient and conserved mechanisms related to nutrition and pathogenesis in bacteria, spore formation in fungi, and signal transduction, protein secretion and antigen presentation in eukaryotes. Furthermore, one of the major causes of drug resistance and chemotherapeutic failure in both cancer and anti-infective therapies is the active movement of compounds across membranes carried out by ABC transporters. Thus, the clinical relevance of ABC transporters is enormous, and the membrane transporters related to chemoresistance are among the best-studied members of the ABC transporter superfamily. As ABC transporter blockers can be used in combination with current drugs to increase their efficacy, the (possible) impact of efflux pump inhibitors is of great clinical interest. The present review summarizes the progress made in recent years in the identification, design, availability, and applicability of ABC transporter blockers in experimental scenarios oriented towards improving the treatment of infectious diseases caused by microorganisms including parasites.     

ABC transporter architecture and mechanism: implications from the crystal structures of BtuCD and BtuF

ABC transporters are ubiquitous membrane proteins that facilitate unidirectional substrate translocation across the lipid bilayer. Over the past five years, new crystal structures have advanced our understanding of how ABC transporters couple adenosine triphosphate (ATP) hydrolysis to substrate transport. In the following, we will briefly review the results of these structural investigations and outline their mechanistic implications.     

Transport of lipids by ABC proteins: Interactions and implications for cellular toxicity, viability and function

Members of the ATP-binding cassette (ABC) family of membrane-bound transporters are involved in multiple aspects of transport and redistribution of various lipids and their conjugates. Most ABC transporters localize to the plasma membrane; some are associated with liquid-ordered cholesterol-/sphingolipid-rich microdomains, and to a lesser extent the membranes of the Golgi and endoplasmic reticulum. Hence, ABC transporters are well placed to regulate plasma membrane lipid composition and the efflux and redistribution of structural phospholipids and sphingolipids during periods of cellular stress and recovery. ABC transporters can also modulate cellular sensitivity to extrinsic pro-apoptotic signals through regulation of sphingomyelin-ceramide biosynthesis and metabolism. The functionality of ABC transporters is, in turn, modulated by the lipid content of the microdomains in which they reside. Cholesterol, a major membrane microdomain component, is not only a substrate of several ABC transporters, but also regulates ABC activity through its effects on microdomain structure. Several important bioactive lipid mediators and toxic lipid metabolites are also effluxed by ABC transporters. In this review, the complex interactions between ABC transporters and their lipid/sterol substrates will be discussed and analyzed in the context of their relevance to cellular function, toxicity and apoptosis.     

Three-dimensional structure by cryo-electron microscopy of YvcC, an homodimeric ATP-binding cassette transporter from Bacillus subtilis

YvcC, a multidrug transporter from Bacillus subtilis, is a member of the ATP-binding cassette superfamily, highly homologous to each half of human multidrug-resistance P-glycoprotein and to several other bacterial half-ABC transporters. Here, the purified recombinant histidine-tagged YvcC has been reconstituted into a lipid bilayer. Controlled and partial detergent removal from YvcC-lipid micelles allowed the production of particularly interesting lipid-detergent-YvcC ring-shaped particles, about 40 nm in diameter, well suited for single particle analysis by cryo-electron microscopy. Furthermore, binding of these histidine-tagged ring-shaped particles to lipid layers functionalized with a Ni(2+)-chelating head group generated a preferential perpendicular orientation, eliminating the missing cone in the final three-dimensional reconstruction. From such analysis, a computed volume has been determined to 2.5 nm resolution giving a detailed insight into the structural organization of this half-ABC transporter within a membrane. The repetitive unit in the ring-shaped particles is consistent with a homodimeric organization of YvcC. Each subunit was composed of three domains: a 5 nm height transmembrane region, a stalk of about 4 nm in height and 2 nm in diameter, and a cytoplasmic lobe of about 5-6 nm in diameter. The latest domain, which fitted with the reported X-ray structure of HisP, was identified as the nucleotide-binding domain (NBD). The 3D reconstruction of the YvcC homodimer well compared with the very recent X-ray crystallographic data on the MsbA homodimer from Escherichia coli, supporting the existence of a central open chamber between the two subunits constituting the homodimer. In addition, the 3D reconstruction of YvcC embedded in a membrane revealed an asymmetric organization of the two NBDs sites within the homodimer, as well as a dimeric interaction between two homodimers.     

Multidrug resistance in parasites: ABC transporters, P-glycoproteins and molecular modelling

Parasitic diseases, caused by protozoa, helminths and arthropods, rank among the most important problems in human and veterinary medicine, and in agriculture, leading to debilitating sicknesses and loss of life. In the absence of vaccines and with the general failure of vector eradication programs, drugs are the main line of defence, but the newest drugs are being tracked by the emergence of resistance in parasites, sharing ominous parallels with multidrug resistance in bacterial pathogens. Any of a number of mechanisms will elicit a drug resistance phenotype in parasites, including: active efflux, reduced uptake, target modification, drug modification, drug sequestration, by-pass shunting, or substrate competition. The role of ABC transporters in parasitic multidrug resistance mechanisms is being subjected to more scrutiny, due in part to the established roles of certain ABC transporters in human diseases, and also to an increasing portfolio of ABC transporters from parasite genome sequencing projects. For example, over 100 ABC transporters have been identified in the Escherichia coli genome, but to date only about 65 in all parasitic genomes. Long established laboratory investigations are now being assisted by molecular biology, bioinformatics, and computational modelling, and it is in these areas that the role of ABC transporters in parasitic multidrug resistance mechanisms may be defined and put in perspective with that of other proteins. We discuss ABC transporters in parasites, and conclude with an example of molecular modelling that identifies a new interaction between the structural domains of a parasite P-glycoprotein.     

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.     

MED: a new non-supervised gene prediction algorithm for bacterial and archaeal genomes

Background  

Despite a remarkable success in the computational prediction of genes in Bacteria and Archaea, a lack of comprehensive understanding of prokaryotic gene structures prevents from further elucidation of differences among genomes. It continues to be interesting to develop new ab initio algorithms which not only accurately predict genes, but also facilitate comparative studies of prokaryotic genomes.     

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.     

Cholesterol: Coupling between membrane microenvironment and ABC transporter activity

Lipid composition of biological membranes is closely related to the function of the ATP-binding cassette (ABC) transporter P-Glycoprotein (Pgp). Herein, we studied how membrane physico-chemical properties affect Pgp-activity. We effectively modulated the cellular cholesterol content using methyl-beta-cyclodextrin (MbetaCD) and MbetaCD-cholesterol-inclusion complex. Pgp was not liberated from the plasma membrane during cholesterol modulation and functional inhibition of Pgp was related to varying cholesterol levels in the plasma membrane. Our data indicate that membrane fluidity does not solely account for cholesterol dependent modifications of Pgp-activity. Therefore, we isolated lipid rafts and examined distinct membrane microdomains. Both depletion and cholesterol enrichment induces a disassembly of lipid rafts. In cholesterol-depleted cell membranes a shift in the Pgp localisation to detergent soluble fractions was observed. Enrichment of membrane cholesterol changed lipid raft distribution but not the localisation of Pgp. From our data we conclude that Pgp-transport capacity depends on accurate lipid raft properties.     

ATP-binding cassette transporter ABCA4: Molecular properties and role in vision and macular degeneration

ABCA4, also known as ABCR or the rim protein, is a member of the ABCA subfamily of ATP binding cassette (ABC) transporters expressed in vertebrate rod and cone photoreceptor cells and localized to outer segment disk membranes. ABCA4 is organized in two tandem halves, each consisting of a transmembrane segment followed successively by a large exocytoplasmic domain, a multispanning membrane domain, and a nucleotide-binding domain. Over 400 mutations in ABCA4 have been linked to Stargardt macular degeneration and related retinal degenerative diseases that cause severe vision loss in affected individuals. Direct binding studies and ATPase activation measurements have identified N-retinylidene-phosphatidylethanolamine, a product generated from the photobleaching of rhodopsin, as the substrate for ABCA4. Mice deficient in ABCA4 accumulate phosphatidylethanolamine, all-trans retinal, and N-retinylidene-phosphatidylethanolamine in photoreceptors and the diretinal pyridinium compound A2E in retinal pigment epithelial cells. On the basis of these studies, ABCA4 is proposed to actively transport or flip N-retinylidene-phosphatidylethanolamine from the lumen to the cytoplasmic side of disc membranes following the photobleaching of rhodopsin. This transport activity insures that retinoids do not accumulate in disc membranes. Disease-linked mutations in ABCA4 that result in diminished transport activity lead to an accumulation of all-trans retinal and N-retinylidene-PE in disc membranes which react to produce A2E precursors. A2E progressively accumulates as lipofuscin deposits in retinal pigment epithelial cells as a result of phagocytosis of outer segment discs. A2E and photo-oxidation products cause RPE cell death and consequently photoreceptor degeneration resulting in a loss in vision in individuals with Stargardt macular degeneration and other retinal degenerative diseases associated with mutations in ABCA4.     

Bird mitochondrial gene order: insight from 3 warbler mitochondrial genomes

Two main gene orders exist in birds: the ancestral gene order and the remnant control region (CR) 2 gene order. These gene orders differ by the presence of 1 or 2 copies of the CR, respectively. Among songbirds, Oscines were thought to follow the ancestral gene order, with the exception of the lyrebird and Phylloscopus warblers. Here, we determined the complete mitochondrial genome sequence of 3 non-Phylloscopus warblers species and found that the blackcap (Sylvia atricapilla) and the reed warbler (Acrocephalus scirpaceus) have 2 almost identical copies of the CR, whereas the eastern orphean warbler (Sylvia crassirostris) follows the remnant CR 2 gene order. Our results contradict previous studies suggesting that Acrocephalus and most sylvioid warblers exhibit the ancestral gene order. We were able to trace this contradiction to a misidentification of gene order from polymerase chain reaction length determination. We thus suggest that passerine gene order evolution needs to be revised.     

Nitensidine A,a guanidine alkaloid from Pterogyne nitens,is a novel substrate for human ABC transporter ABCB1

The Pterogyne nitens (Fabaceae) tree, native to South America, has been found to produce guanidine alkaloids as well as bioactive flavonols such as kaempferol, quercetin, and rutin. In the present study, we examined the possibility of interaction between human ATP-binding cassette (ABC) transporter ABCB1 and four guanidine alkaloids isolated from P. nitens (i.e., galegine, nitensidine A, pterogynidine, and pterogynine) using human T cell lymphoblast-like leukemia cell line CCRF-CEM and its multi-drug resistant (MDR) counterpart CEM/ADR5000. In XTT assays, CEM/ADR5000 cells were resistant to the four guanidine alkaloids compared to CCRF-CEM cells, although the four guanidine alkaloids exhibited some level of cytotoxicity against both CCRF-CEM and CEM/ADR5000 cells. In ATPase assays, three of the four guanidine alkaloids were found to stimulate the ATPase activity of ABCB1. Notably, nitensidine A was clearly found to stimulate the ATPase activity of ABCB1 as strongly as the control drug, verapamil. Furthermore, the cytotoxic effect of nitensidine A on CEM/ADR5000 cells was synergistically enhanced by verapamil. Nitensidine A inhibited the extrusion of calcein by ABCB1. In the present study, the possibility of interaction between ABCB1 and two synthetic nitensidine A analogs (nitensidine AT and AU) were examined to gain insight into the mechanism by which nitensidine A stimulates the ATPase activity of ABCB1. The ABCB1-dependent ATPase activity stimulated by nitensidine A was greatly reduced by substituting sulfur (S) or oxygen (O) for the imino nitrogen atom (N) in nitensidine A. Molecular docking studies on human ABCB1 showed that, guanidine alkaloids from P. nitens dock to the same binding pocket as verapamil. Nitensidine A and its analogs exhibit similar binding energies to verapamil. Taken together, this research clearly indicates that nitensidine A is a novel substrate for ABCB1. The present results also suggest that the number, binding site, and polymerization degree of the isoprenyl moiety in the guanidine alkaloids and the imino nitrogen atom cooperatively contribute to their stimulation of ABCB1's ATPase activity.     

Conformational changes in MetNI: steered molecular dynamic studies of the methionine ABC transporter with and without substrates

ATP-binding cassette (ABC) transporters are integral membrane proteins that utilised energy from ATP hydrolysis to translocate substrates across the membrane. In addition to the common nucleotide-binding domains (NBDs) and transmembrane domains (TMDs), the methionine ABC transporter has C-terminal regulatory domains (C2 domains) that belong to ACT protein family. When the amount of methionine in the cell is high, the transport stops. This phenomenon is called trans-inhibition. To understand how a trans-inhibited protein returns to an uninhibited, resting state, we performed steered molecular dynamic simulations with and without the substrates. From the simulations, we observed some important conformational changes in the whole ABC transporter, including the constriction in the translocation pathway in the TMDs and approach of the NBDs. However, the C2 domains behaved differently in two types of the simulations. These findings might help to explain the relationship of the conformational changes of the C2 domains with the rearrangements of the NBDs or TMDs, and provide a way to understand the trans-inhibition from an opposite direction.     

Statistical analysis of complete bacterial genomes: Avoidance of palindromes and restriction-modification systems

Avoidance of 4-, 5-, and 6-letter palindromes is observed in many prokaryotic genomes. A large fraction of such palindromes is formed by restriction sites of the species itself or a closely related species. One possible reason for that is the horizontal transfer of genes encoding restriction-modification systems. In organisms isolated from the action of such systems (e.g., in Mycoplasma), palindromes are not avoided. The general tendencies in preferences and avoidance of palindromes were studied for 33 available prokaryotic genomes. The results obtained provide additional insight into the relationships within and between taxonomic groups.