The substrate-binding domains of the osmoregulatory ABC importer OpuA transiently interact.

Bacteria utilize various strategies to prevent internal dehydration during hypertonic stress. A common approach to countering the effects of the stress is to import compatible solutes such as glycine betaine, leading to simultaneous passive water fluxes following the osmotic gradient. OpuA from Lactococcus lactis is a type I ABC-importer that uses two substrate-binding domains (SBDs) to capture extracellular glycine betaine and deliver the substrate to the transmembrane domains for subsequent transport. OpuA senses osmotic stress via changes in the internal ionic strength and is furthermore regulated by the 2nd messenger cyclic-di-AMP. We now show, by means of solution-based single-molecule FRET and analysis with multi-parameter photon-by-photon hidden Markov modeling, that the SBDs transiently interact in an ionic strength-dependent manner. The smFRET data are in accordance with the apparent cooperativity in transport and supported by new cryo-EM data of OpuA. We propose that the physical interactions between SBDs and cooperativity in substrate delivery are part of the transport mechanism.

An ATP13A1-assisted topogenesis pathway for folding multi-spanning membrane proteins.

Many multi-spanning membrane proteins contain poorly hydrophobic transmembrane domains (pTMDs) protected from phospholipid in mature structure. Nascent pTMDs are difficult for translocon to recognize and insert. How pTMDs are discerned and packed into mature, muti-spanning configuration remains unclear. Here, we report that pTMD elicits a post-translational topogenesis pathway for its recognition and integration. Using six-spanning protein adenosine triphosphate-binding cassette transporter G2 (ABCG2) and cultured human cells as models, we show that ABCG2's pTMD2 can pass through translocon into the endoplasmic reticulum (ER) lumen, yielding an intermediate with inserted yet mis-oriented downstream TMDs. After translation, the intermediate recruits P5A-ATPase ATP13A1, which facilitates TMD re-orientation, allowing further folding and the integration of the remaining lumen-exposed pTMD2. Depleting ATP13A1 or disrupting pTMD-characteristic residues arrests intermediates with mis-oriented and exposed TMDs. Our results explain how a "difficult" pTMD is co-translationally skipped for insertion and post-translationally buried into the final correct structure at the late folding stage to avoid excessive lipid exposure.

Molecular insights into capsular polysaccharide secretion.

Capsular polysaccharides (CPSs) fortify the cell boundaries of many commensal and pathogenic bacteria1. Through the ABC-transporter-dependent biosynthesis pathway, CPSs are synthesized intracellularly on a lipid anchor and secreted across the cell envelope by the KpsMT ABC transporter associated with the KpsE and KpsD subunits1,2. Here we use structural and functional studies to uncover crucial steps of CPS secretion in Gram-negative bacteria. We show that KpsMT has broad substrate specificity and is sufficient for the translocation of CPSs across the inner bacterial membrane, and we determine the cell surface organization and localization of CPSs using super-resolution fluorescence microscopy. Cryo-electron microscopy analyses of the KpsMT-KpsE complex in six different states reveal a KpsE-encaged ABC transporter, rigid-body conformational rearrangements of KpsMT during ATP hydrolysis and recognition of a glycolipid inside a membrane-exposed electropositive canyon. In vivo CPS secretion assays underscore the functional importance of canyon-lining basic residues. Combined, our analyses suggest a molecular model of CPS secretion by ABC transporters.

New Paralogs of the Heliothis virescens ABCC2 Transporter as Potential Receptors for Bt Cry1A Proteins.

The ATP-binding cassette (ABC) transporters are a superfamily of membrane proteins. These active transporters are involved in the export of different substances such as xenobiotics. ABC transporters from subfamily C (ABCC) have also been described as functional receptors for different insecticidal proteins from Bacillus thuringiensis (Bt) in several lepidopteran species. Numerous studies have characterized the relationship between the ABCC2 transporter and Bt Cry1 proteins. Although other ABCC transporters sharing structural and functional similarities have been described, little is known of their role in the mode of action of Bt proteins. For Heliothis virescens, only the ABCC2 transporter and its interaction with Cry1A proteins have been studied to date. Here, we have searched for paralogs to the ABCC2 gene in H. virescens, and identified two new ABC transporter genes: HvABCC3 and HvABCC4. Furthermore, we have characterized their gene expression in the midgut and their protein topology, and compared them with that of ABCC2. Finally, we discuss their possible interaction with Bt proteins by performing protein docking analysis.

Structure and function of the Arabidopsis ABC transporter ABCB19 in brassinosteroid export.

Brassinosteroids are steroidal phytohormones that regulate plant development and physiology, including adaptation to environmental stresses. Brassinosteroids are synthesized in the cell interior but bind receptors at the cell surface, necessitating a yet to be identified export mechanism. Here, we show that a member of the ATP-binding cassette (ABC) transporter superfamily, ABCB19, functions as a brassinosteroid exporter. We present its structure in both the substrate-unbound and the brassinosteroid-bound states. Bioactive brassinosteroids are potent activators of ABCB19 ATP hydrolysis activity, and transport assays showed that ABCB19 transports brassinosteroids. In Arabidopsis thaliana, ABCB19 and its close homolog, ABCB1, positively regulate brassinosteroid responses. Our results uncover an elusive export mechanism for bioactive brassinosteroids that is tightly coordinated with brassinosteroid signaling.

Molecular basis for substrate transport of Mycobacterium tuberculosis ABC importer DppABCD.

The type I adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter DppABCD is believed to be responsible for the import of exogenous heme as an iron source into the cytoplasm of the human pathogen Mycobacterium tuberculosis (Mtb). Additionally, this system is also known to be involved in the acquisition of tri- or tetra-peptides. Here, we report the cryo-electron microscopy structures of the dual-function Mtb DppABCD transporter in three forms, namely, the apo, substrate-bound, and ATP-bound states. The apo structure reveals an unexpected and previously uncharacterized assembly mode for ABC importers, where the lipoprotein DppA, a cluster C substrate-binding protein (SBP), stands upright on the translocator DppBCD primarily through its hinge region and N-lobe. These structural data, along with biochemical studies, reveal the assembly of DppABCD complex and the detailed mechanism of DppABCD-mediated transport. Together, these findings provide a molecular roadmap for understanding the transport mechanism of a cluster C SBP and its translocator.

Cardiolipin Regulates the Activity of the Mitochondrial ABC Transporter ABCB10.

The ATP-binding cassette (ABC) transporter ABCB10 resides in the inner membrane of mitochondria and is implicated in erythropoiesis. Mitochondria from different cell types share some specific characteristics, one of which is the high abundance of cardiolipin. Although previous studies have provided insight into ABCB10, the affinity and selectivity of this transporter toward lipids, particularly those found in the mitochondria, remain poorly understood. Here, native mass spectrometry is used to directly monitor the binding events of lipids to human ABCB10. The results reveal that ABCB10 binds avidly to cardiolipin with an affinity significantly higher than that of other phospholipids. The first three binding events of cardiolipin display positive cooperativity, which is suggestive of specific cardiolipin-binding sites on ABCB10. Phosphatidic acid is the second-best binder of the lipids investigated. The bulk lipids, phosphatidylcholine and phosphatidylethanolamine, display the weakest binding affinity for ABCB10. Other lipids bind ABCB10 with a similar affinity. Functional assays show that cardiolipin regulates the ATPase activity of ABCB10 in a dose-dependent fashion. ATPase activity of ABCB10 was also impacted in the presence of other lipids but to a lesser extent than cardiolipin. Taken together, ABCB10 has a high binding affinity for cardiolipin, and this lipid also regulates the ATPase activity of the transporter.

Elucidating the structural and functional prophecy of the Rv2326c gene, an ABC transporter of Mycobacterium tuberculosis H37Rv through computational approach.

Tuberculosis is a fatal disease caused by Mycobacterium tuberculosis. M. tuberculosis becoming drug-resistant day by day, necessitating to know the mechanism behind the drug resistance and how to overcome this deadly malady. Drug resistance and reduced drug bioavailability are caused by a class of transporter proteins called the ATP-binding cassette (ABC) transporters, which pump a range of medicines out of cells at the price of ATP hydrolysis. By using computational approaches, we tried to elaborate the probable function of the Rv2326c gene of M. tuberculosis, perhaps involved in drug resistance mechanism. The presence of the signature motif of ABC transporters (LSGGELQRLALAAAL and LSGGQMRRVVLAGLL) and ATP binding motif (GXXXXGKT and GXXXXGKS) in the protein sequence signifying its importance in the ATP binding and transportation of molecules. Further, this manuscript elaborated about tertiary structure and validation, functional category, localization, phosphorylation site prediction, mutational analysis of conserved motifs. Ligand docking study shows the highest affinity with ATP than GTP justified its function as an ATP binding protein. The Rv2326c protein is present in the inner membrane and working as an ATP binding protein and might be playing a dynamic role in transportation. In this study, we found that Rv2326c protein might be working as an ABC transporter by which the drugs and other molecules are imported or exported into the bacterium. As a result, the current study provides a means to better understand its normal functioning and basic biology, which can help in the development of novel therapeutic targeting approaches for Rv2326c protein.

Genome-wide identification and characterization of ABC transporter superfamily in the legume Cajanus cajan.

Plant ATP-binding cassette (ABC) protein family is the largest multifunctional highly conserved protein superfamily that transports diverse substrates across biological membranes by the hydrolysis of ATP and is also the part of the several other biological processes like cellular detoxification, growth and development, stress biology, and signaling processes. In the agriculturally important legume crop Cajanus cajan, a genome-wide identification and characterization of the ABC gene family was carried out. A total of 159 ABC genes were identified that belong to eight canonical classes CcABCA to CcABCG and CcABCI based on the phylogenetic analysis. The number of genes was highest in CcABCG followed by CcABCC and CcABCB class. A total of 85 CcABC genes were found on 11 chromosomes and 74 were found on scaffold. Tandem duplication was the major driver of CcABC gene family expansion. The dN/dS ratio revealed the purifying selection. The phylogenetic analysis revealed class-specific eight superclades which reflect their functional importance. The largest clade was found to be CcABCG which reflects their functional significance. CcABC proteins were mainly basic in nature and found to be localized in the plasma membrane. The secondary structure prediction revealed the dominance of α-helix. The canonical transmembrane and nucleotide binding domain, signature motif LSSGQ, Walker A, Walker B region, and Q loop were also identified. A class-specific exon-intron pattern was also observed. In addition to core elements, different cis-acting regulatory elements like stress, hormone, and cellular responsive were also identified. Expression profiling of CcABC genes at various developmental stages of different anatomical tissues was performed and it was noticed that CcABCF3, CcABCF4, CcABCF5, CcABCG66, and CcABCI3 had the highest expression. The results of the current study endow us with the further functional analysis of Cajanus ABC in the future.

Time-Resolved Mn2+ -NO and NO-NO Distance Measurements Reveal That Catalytic Asymmetry Regulates Alternating Access in an ABC Transporter.

ATP-binding cassette (ABC) transporters shuttle diverse substrates across biological membranes. Transport is often achieved through a transition between an inward-facing (IF) and an outward-facing (OF) conformation of the transmembrane domains (TMDs). Asymmetric nucleotide-binding sites (NBSs) are present among several ABC subfamilies and their functional role remains elusive. Here we addressed this question using concomitant NO-NO, Mn2+ -NO, and Mn2+ -Mn2+ pulsed electron-electron double-resonance spectroscopy of TmrAB in a time-resolved manner. This type-IV ABC transporter undergoes a reversible transition in the presence of ATP with a significantly faster forward transition. The impaired degenerate NBS stably binds Mn2+ -ATP, and Mn2+ is preferentially released at the active consensus NBS. ATP hydrolysis at the consensus NBS considerably accelerates the reverse transition. Both NBSs fully open during each conformational cycle and the degenerate NBS may regulate the kinetics of this process.

FtsE, the Nucleotide Binding Domain of the ABC Transporter Homolog FtsEX, Regulates Septal PG Synthesis in E. coli.

The peptidoglycan (PG) layer, a crucial component of the tripartite E.coli envelope, is required to maintain cellular integrity, protecting the cells from mechanical stress resulting from intracellular turgor pressure. Thus, coordinating synthesis and hydrolysis of PG during cell division (septal PG) is crucial for bacteria. The FtsEX complex directs septal PG hydrolysis through the activation of amidases; however, the mechanism and regulation of septal PG synthesis are unclear. In addition, how septal PG synthesis and hydrolysis are coordinated has remained unclear. Here, we have shown that overexpression of FtsE leads to a mid-cell bulging phenotype in E.coli, which is different from the filamentous phenotype observed during overexpression of other cell division proteins. Silencing of the common PG synthesis genes murA and murB reduced bulging, confirming that this phenotype is due to excess PG synthesis. We further demonstrated that septal PG synthesis is independent of FtsE ATPase activity and FtsX. These observations and previous results suggest that FtsEX plays a role during septal PG hydrolysis, whereas FtsE alone coordinates septal PG synthesis. Overall, our study findings support a model in which FtsE plays a role in coordinating septal PG synthesis with bacterial cell division. IMPORTANCE The peptidoglycan (PG) layer is an essential component of the E.coli envelope that is required to maintain cellular shape and integrity. Thus, coordinating PG synthesis and hydrolysis at the mid-cell (septal PG) is crucial during bacterial division. The FtsEX complex directs septal PG hydrolysis through the activation of amidases; however, its role in regulation of septal PG synthesis is unclear. Here, we demonstrate that overexpression of FtsE in E.coli leads to a mid-cell bulging phenotype due to excess PG synthesis. This phenotype was reduced upon silencing of common PG synthesis genes murA and murB. We further demonstrated that septal PG synthesis is independent of FtsE ATPase activity and FtsX. These observations suggest that the FtsEX complex plays a role during septal PG hydrolysis, whereas FtsE alone coordinates septal PG synthesis. Our study indicates that FtsE plays a role in coordinating septal PG synthesis with bacterial cell division.

Biosensor for Multimodal Characterization of an Essential ABC Transporter for Next-Generation Antibiotic Research.

As the threat of antibiotic resistance increases, there is a particular focus on developing antimicrobials against pathogenic bacteria whose multidrug resistance is especially entrenched and concerning. One such target for novel antimicrobials is the ATP-binding cassette (ABC) transporter MsbA that is present in the plasma membrane of Gram-negative pathogenic bacteria where it is fundamental to the survival of these bacteria. Supported lipid bilayers (SLBs) are useful in monitoring membrane protein structure and function since they can be integrated with a variety of optical, biochemical, and electrochemical techniques. Here, we form SLBs containing Escherichia coli MsbA and use atomic force microscopy (AFM) and structured illumination microscopy (SIM) as high-resolution microscopy techniques to study the integrity of the SLBs and incorporated MsbA proteins. We then integrate these SLBs on microelectrode arrays (MEA) based on the conducting polymer poly(3,4-ethylenedioxy-thiophene) poly(styrene sulfonate) (PEDOT:PSS) using electrochemical impedance spectroscopy (EIS) to monitor ion flow through MsbA proteins in response to ATP hydrolysis. These EIS measurements can be correlated with the biochemical detection of MsbA-ATPase activity. To show the potential of this SLB approach, we observe not only the activity of wild-type MsbA but also the activity of two previously characterized mutants along with quinoline-based MsbA inhibitor G907 to show that EIS systems can detect changes in ABC transporter activity. Our work combines a multitude of techniques to thoroughly investigate MsbA in lipid bilayers as well as the effects of potential inhibitors of this protein. We envisage that this platform will facilitate the development of next-generation antimicrobials that inhibit MsbA or other essential membrane transporters in microorganisms.

Structure and Mechanism of Human ABC Transporters.

ABC transporters are essential for cellular physiology. Humans have 48 ABC genes organized into seven distinct families. Of these genes, 44 (in five distinct families) encode for membrane transporters, of which several are involved in drug resistance and disease pathways resulting from transporter dysfunction. Over the last decade, advances in structural biology have vastly expanded our mechanistic understanding of human ABC transporter function, revealing details of their molecular arrangement, regulation, and interactions, facilitated in large part by advances in cryo-EM that have rendered hitherto inaccessible targets amenable to high-resolution structural analysis. As a result, experimentally determined structures of multiple members of each of the five families of ABC transporters in humans are now available. Here we review this recent progress, highlighting the physiological relevance of human ABC transporters and mechanistic insights gleaned from their direct structure determination. We also discuss the impact and limitations of model systems and structure prediction methods in understanding human ABC transporters and discuss current challenges and future research directions.

A conserved WXXE motif is an apical delivery determinant of ABC transporter C subfamily isoforms.

ATP-binding cassette transporter isoform C7 (ABCC7), also designated as cystic fibrosis transmembrane conductance regulator (CFTR), is exclusively targeted to the apical plasma membrane of polarized epithelial cells. Although the apical localization of ABCC7 in epithelia is crucial for the Cl- excretion into lumens, the mechanism regulating its apical localization is poorly understood. In the present study, an apical localization determinant was identified in the N-terminal 80-amino acid long cytoplasmic region of ABCC7 (NT80). In HepG2 cells, overexpression of NT80 significantly disturbed the apical expression of ABCC7 in a competitive manner, suggesting the presence of a sorting determinant in this region. Deletion analysis identified a potential sorting information within a 20-amino acid long peptide (aa 41-60) of NT80. Alanine scanning mutagenesis of this region in full-length ABCC7 further narrowed down the apical localization determinant to four amino acids, W57DRE60. This WDRE sequence was conserved among vertebrate ABCC7 orthologs. Site-directed mutagenesis showed that W57 and E60 were critical for the apical expression of ABCC7, confirming a novel apical sorting determinant of ABCC7. Furthermore, a WXXE motif (tryptophan and glutamic acid residues with two-amino acid spacing) was found to be conserved among the N-terminal regions of apically localized ABCC members with 12-TM configuration. The significance of the WXXE motif was demonstrated for proper trafficking of ABCC4 to the apical plasma membrane.Key words: apical plasma membrane, sorting, ATP-binding cassette transporter, CFTR, MRP4.

Delayed Diagnosis of Chronic Necrotizing Granulomatous Skin Lesions due to TAP2 Deficiency.

Major histocompatibility complex class I (MHC-I) deficiency, also known as bare lymphocyte syndrome type 1 (BLS-1), is a rare autosomal recessively inherited immunodeficiency disorder with remarkable clinical and biological heterogeneity. Transporter associated with antigen processing (TAP) is a member of the ATP-binding cassette superfamily of transporters and consists of two subunits, TAP1 or TAP2. Any defect resulting from a mutation or deletion of these two subunits may adversely affect the peptide translocation in the endoplasmic reticulum, which is an important process for properly assembling MHC-I molecules. To date, only 12 TAP2-deficient patients were reported in the literature. Herein, we described two Iranian cases with 2 and 3 decades of delayed diagnosis of chronic necrotizing granulomatous skin lesions due to TAP2 deficiency without pulmonary involvement. Segregation analysis in family members identified 3 additional homozygous asymptomatic carriers. In both asymptomatic and symptomatic carriers, HLA-I expression was only 4-15% of the one observed in healthy controls. We performed the first deep immunophenotyping in TAP2-deficient patients. While total CD8 T cell counts were normal as previously reported, the patients showed strongly impaired naïve CD8 T cell counts. Mucosal-associated invariant T (MAIT) cells and invariant natural killer T (iNKT) cell counts were increased.

Waste or die: The price to pay to stay alive.

Microorganisms need to constantly exchange with their habitat to capture nutrients and expel toxic compounds. The ATP-binding cassette (ABC) transporters, a family of membrane proteins especially abundant in microorganisms, are at the core of these processes. Due to their extraordinary ability to expel structurally unrelated compounds, some transporters play a protective role in different organisms. Yet, the downside of these multidrug transporters is their entanglement in the resistance to therapeutic treatments. Intriguingly, some multidrug ABC transporters show a high level of ATPase activity, even in the absence of transported substrates. Although this basal ATPase activity might seem a waste, we surmise that this inherent capacity allows multidrug transporters to promptly translocate any bound drug before it penetrates into the cell.

The ABC transporter MsbA adopts the wide inward-open conformation in E. coli cells.

Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance. Unexpectedly, the wide inward-open conformation of MsbA, commonly considered a nonphysiological state, was found to be prominently populated in Escherichia coli cells. Molecular dynamics simulations revealed that extensive lateral portal opening is essential to provide access of its large natural substrate core lipid A to the binding cavity. Our work paves the way to investigate the conformational landscape of membrane proteins in cells.

ATP-Binding Cassette Transporters: Snap-on Complexes?

ATP-binding cassette (ABC) transporters are one of the largest families of membrane proteins in prokaryotic organisms. Much is now understood about the structure of these transporters and many reviews have been written on that subject. In contrast, less has been written on the assembly of ABC transporter complexes and this will be a major focus of this book chapter. The complexes are formed from two cytoplasmic subunits that are highly conserved (in terms of their primary and three-dimensional structures) across the whole family. These ATP-binding subunits give rise to the name of the family. They must assemble with two transmembrane subunits that will typically form the permease component of the transporter. The transmembrane subunits have been found to be surprisingly diverse in structure when the whole family is examined, with seven distinct folds identified so far. Hence nucleotide-binding subunits appear to have been bolted on to a variety of transmembrane platforms during evolution, leading to a greater variety in function. Furthermore, many importers within the family utilise a further external substrate-binding component to trap scarce substrates and deliver them to the correct permease components. In this chapter, we will discuss whether assembly of the various ABC transporter subunits occurs with high fidelity within the crowded cellular environment and whether promiscuity in assembly of transmembrane and cytoplasmic components can occur. We also discuss the new AlphaFold protein structure prediction tool which predicts a new type of transmembrane domain fold within the ABC transporters that is associated with cation exporters of bacteria and plants.

Regulation of Lytic Machineries by the FtsEX Complex in the Bacterial Divisome.

The essential membrane complex FtsE/FtsX (FtsEX), belonging to the ABC transporter superfamily and widespread among bacteria, plays a relevant function in some crucial cell wall remodeling processes such as cell division, elongation, or sporulation. FtsEX plays a double role by recruiting proteins to the divisome apparatus and by regulating lytic activity of the cell wall hydrolases required for daughter cell separation. Interestingly, FtsEX does not act as a transporter but uses the ATPase activity of FtsE to mechanically transmit a signal from the cytosol, through the membrane, to the periplasm that activates the attached hydrolases. While the complete molecular details of such mechanism are not yet known, evidence has been recently reported that clarify essential aspects of this complex system. In this chapter we will present recent structural advances on this topic. The three-dimensional structure of FtsE, FtsX, and some of the lytic enzymes or their cognate regulators revealed an unexpected scenario in which a delicate set of intermolecular interactions, conserved among different bacterial genera, could be at the core of this regulatory mechanism providing exquisite control in both space and time of this central process to assist bacterial survival.

ATP-Binding Cassette G Transporters and Their Multiple Roles Especially for Male Fertility in Arabidopsis, Rice and Maize.

ATP-binding cassette subfamily G (ABCG) transporters are extensive in plants and play essential roles in various processes influencing plant fitness, but the research progress varies greatly among Arabidopsis, rice and maize. In this review, we present a consolidated nomenclature and characterization of the whole 51 ABCG transporters in maize, perform a phylogenetic analysis and classification of the ABCG subfamily members in maize, and summarize the latest research advances in ABCG transporters for these three plant species. ABCG transporters are involved in diverse processes in Arabidopsis and rice, such as anther and pollen development, vegetative and female organ development, abiotic and biotic stress response, and phytohormone transport, which provide useful clues for the functional investigation of ABCG transporters in maize. Finally, we discuss the current challenges and future perspectives for the identification and mechanism analysis of substrates for plant ABCG transporters. This review provides a basic framework for functional research and the potential application of ABCG transporters in multiple plants, including maize.