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.

Probing the stability and interdomain interactions in the ABC transporter OpuA using single-molecule optical tweezers.

Transmembrane transporter proteins are essential for maintaining cellular homeostasis and, as such, are key drug targets. Many transmembrane transporter proteins are known to undergo large structural rearrangements during their functional cycles. Despite the wealth of detailed structural and functional data available for these systems, our understanding of their dynamics and, consequently, how they function is generally limited. We introduce an innovative approach that enables us to directly measure the dynamics and stability of interdomain interactions of transmembrane proteins using optical tweezers. Focusing on the osmoregulatory ATP-binding cassette transporter OpuA from Lactococcus lactis, we examine the mechanical properties and potential interactions of its substrate-binding domains. Our measurements are performed in lipid nanodiscs, providing a native-mimicking environment for the transmembrane protein. The technique provides high spatial and temporal resolution and allows us to study the functionally relevant motions and interdomain interactions of individual transmembrane transporter proteins in real time in a lipid bilayer.

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.

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.

The ABC toxin complex from Yersinia entomophaga can package three different cytotoxic components expressed from distinct genetic loci in an unfolded state: the structures of both shell and cargo.

Bacterial ABC toxin complexes (Tcs) comprise three core proteins: TcA, TcB and TcC. The TcA protein forms a pentameric assembly that attaches to the surface of target cells and penetrates the cell membrane. The TcB and TcC proteins assemble as a heterodimeric TcB-TcC subcomplex that makes a hollow shell. This TcB-TcC subcomplex self-cleaves and encapsulates within the shell a cytotoxic `cargo' encoded by the C-terminal region of the TcC protein. Here, we describe the structure of a previously uncharacterized TcC protein from Yersinia entomophaga, encoded by a gene at a distant genomic location from the genes encoding the rest of the toxin complex, in complex with the TcB protein. When encapsulated within the TcB-TcC shell, the C-terminal toxin adopts an unfolded and disordered state, with limited areas of local order stabilized by the chaperone-like inner surface of the shell. We also determined the structure of the toxin cargo alone and show that when not encapsulated within the shell, it adopts an ADP-ribosyltransferase fold most similar to the catalytic domain of the SpvB toxin from Salmonella typhimurium. Our structural analysis points to a likely mechanism whereby the toxin acts directly on actin, modifying it in a way that prevents normal polymerization.

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.

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.

Purification and characterization of Cdr1, the drug-efflux pump conferring azole resistance in Candida species.

Candida albicans and C. glabrata express exporters of the ATP-binding cassette (ABC) superfamily and address them to their plasma membrane to expel azole antifungals, which cancels out their action and allows the yeast to become multidrug resistant (MDR). In a way to understand this mechanism of defense, we describe the purification and characterization of Cdr1, the membrane ABC exporter mainly responsible for such phenotype in both species. Cdr1 proteins were functionally expressed in the baker yeast, tagged at their C-terminal end with either a His-tag for the glabrata version, cgCdr1-His, or a green fluorescent protein (GFP) preceded by a proteolytic cleavage site for the albicans version, caCdr1-P-GFP. A membrane Cdr1-enriched fraction was then prepared to assay several detergents and stabilizers, probing their level of extraction and the ATPase activity of the proteins as a functional marker. Immobilized metal-affinity and size-exclusion chromatographies (IMAC, SEC) were then carried out to isolate homogenous samples. Overall, our data show that although topologically and phylogenetically close, both proteins display quite distinct behaviors during the extraction and purification steps, and qualify cgCdr1 as a good candidate to characterize this type of proteins for developing future inhibitors of their azole antifungal efflux activity.

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.

Structure of an endogenous mycobacterial MCE lipid transporter.

To replicate inside macrophages and cause tuberculosis, Mycobacterium tuberculosis must scavenge a variety of nutrients from the host1,2. The mammalian cell entry (MCE) proteins are important virulence factors in M. tuberculosis1,3, where they are encoded by large gene clusters and have been implicated in the transport of fatty acids4-7 and cholesterol1,4,8 across the impermeable mycobacterial cell envelope. Very little is known about how cargos are transported across this barrier, and it remains unclear how the approximately ten proteins encoded by a mycobacterial mce gene cluster assemble to transport cargo across the cell envelope. Here we report the cryo-electron microscopy (cryo-EM) structure of the endogenous Mce1 lipid-import machine of Mycobacterium smegmatis-a non-pathogenic relative of M. tuberculosis. The structure reveals how the proteins of the Mce1 system assemble to form an elongated ABC transporter complex that is long enough to span the cell envelope. The Mce1 complex is dominated by a curved, needle-like domain that appears to be unrelated to previously described protein structures, and creates a protected hydrophobic pathway for lipid transport across the periplasm. Our structural data revealed the presence of a subunit of the Mce1 complex, which we identified using a combination of cryo-EM and AlphaFold2, and name LucB. Our data lead to a structural model for Mce1-mediated lipid import across the mycobacterial cell envelope.

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.

Simple purification and characterization of soluble and homogenous ABC-F translation factors from Enterococcus faecium.

The family of ATP-binding cassette F proteins (ABC-F) is mainly made up of cytosolic proteins involved in regulating protein synthesis, and they are often part of a mechanism that confers resistance to ribosome-targeting antibiotics. The existing literature has emphasized the difficulty of purifying these recombinant proteins because of their very low solubility and stability. Here, we describe a rapid and efficient three-step purification procedure that allows for the production of untagged ABC-F proteins from Enterococcus faecium in the heterologous host Escherichia coli. After four purified ABC-F proteins were produced using this protocol, their biological activities were validated by in vitro experiment. In conclusion, our study provides an invaluable tool for obtaining large amounts of untagged and soluble ABC-F proteins that can then be used for in vitro experiments.

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.

Cryo-EM structures of mitochondrial ABC transporter ABCB10 in apo and biliverdin-bound form.

ABCB10, a member of ABC transporter superfamily that locates in the inner membrane of mitochondria, plays crucial roles in hemoglobin synthesis, antioxidative stress and stabilization of the iron transporter mitoferrin-1. Recently, it was found that ABCB10 is a mitochondrial biliverdin exporter. However, the molecular mechanism of biliverdin export by ABCB10 remains elusive. Here we report the cryo-EM structures of ABCB10 in apo (ABCB10-apo) and biliverdin-bound form (ABCB10-BV) at 3.67 Å and 2.85 Å resolution, respectively. ABCB10-apo adopts a wide-open conformation and may thus represent the apo form structure. ABCB10-BV forms a closed conformation and biliverdin situates in a hydrophobic pocket in one protomer and bridges the interaction through hydrogen bonds with the opposing one. We also identify cholesterols sandwiched by BVs and discuss the export dynamics based on these structural and biochemical observations.

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.

Redefining the bacterial Type I protein secretion system.

Type I secretion systems (T1SS) are versatile molecular machines for protein transport across the Gram-negative cell envelope. The archetypal Type I system mediates secretion of the Escherichia coli hemolysin, HlyA. This system has remained the pre-eminent model of T1SS research since its discovery. The classic description of a T1SS is composed of three proteins: an inner membrane ABC transporter, a periplasmic adaptor protein and an outer membrane factor. According to this model, these components assemble to form a continuous channel across the cell envelope, an unfolded substrate molecule is then transported in a one-step mechanism, directly from the cytosol to the extracellular milieu. However, this model does not encapsulate the diversity of T1SS that have been characterized to date. In this review, we provide an updated definition of a T1SS, and propose the subdivision of this system into five subgroups. These subgroups are categorized as T1SSa for RTX proteins, T1SSb for non-RTX Ca2+-binding proteins, T1SSc for non-RTX proteins, T1SSd for class II microcins, and T1SSe for lipoprotein secretion. Although often overlooked in the literature, these alternative mechanisms of Type I protein secretion offer many avenues for biotechnological discovery and application.

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.

Purification and characterization of eukaryotic ATP-dependent transporters homologously expressed in Pichia pastoris for structural studies by cryo-electron microscopy.

Membrane proteins play an essential role in all living organisms. Although there have been numerous efforts in the past to elucidate the structure and function of eukaryotic primary active transporters, knowledge about the majority of these membrane proteins is still minimal. This is often due to their low availability and complex handling. In this study, we homologously expressed three ATP-dependent transport proteins, STE6-2p, NEO1-p, and YPK9-p, in Pichia pastoris and subsequently optimized the solubilization and purification processes. Sequential use of different mild detergents and utilization of hydrophilic matrices in the purification procedure allowed us to obtain all three transporters monodisperse and in high purity, enabling initial structural analysis by cryo-electron microscopy. Using the respective substrates, we determined the specific activity of all target proteins using an ATPase assay. This study opens the door to further functional and structural studies of this pharmacologically important class of membrane proteins.