Primary Active Transport Research Articles

The complete catalog of antimicrobial resistance secondary active transporters in Clostridioides difficile: evolution and drug resistance perspective

Secondary active transporters shuttle substrates across eukaryotic and prokaryotic membranes, utilizing different electrochemical gradients. They are recognized as one of the antimicrobial efflux pumps among pathogens. While primary active transporters within the genome of C. difficile 630 have been completely cataloged, the systematical study of secondary active transporters remains incomplete. Here, we not only identify secondary active transporters but also disclose their evolution and role in drug resistance in C. difficile 630. Our analysis reveals that C. difficile 630 carries 147 secondary active transporters belonging to 27 (super)families. Notably, 50 (34%) of them potentially contribute to antimicrobial resistance (AMR). AMR-secondary active transporters are structurally classified into five (super)families: the p-aminobenzoyl-glutamate transporter (AbgT), drug/metabolite transporter (DMT) superfamily, major facilitator (MFS) superfamily, multidrug and toxic compound extrusion (MATE) family, and resistance-nodulation-division (RND) family. Surprisingly, complete RND genes found in C. difficile 630 are likely an evolutionary leftover from the common ancestor with the diderm. Through protein structure comparisons, we have potentially identified six novel AMR-secondary active transporters from DMT, MATE, and MFS (super)families. Pangenome analysis revealed that half of the AMR-secondary transporters are accessory genes, which indicates an important role in adaptive AMR function rather than innate physiological homeostasis. Gene expression profile firmly supports their ability to respond to a wide spectrum of antibiotics. Our findings highlight the evolution of AMR-secondary active transporters and their integral role in antibiotic responses. This marks AMR-secondary active transporters as interesting therapeutic targets to synergize with other antibiotic activity.

Acid tolerance of lactate-utilizing bacteria of the order Bacteroidales contributes to prevention of ruminal acidosis in goats adapted to a high-concentrate diet.

The rapid accumulation of organic acids, particularly lactate, has been suggested as the main cause of ruminal acidosis (RA) for ruminants fed high-concentrate diets. Previous research has shown that a gradual shift from low-to high-concentrate diets within 4 to 5 weeks effectively reduces the risk for RA. However, the mechanisms remain unknown. In this study, 20 goats were randomly allocated into four groups (n=5) and fed with a diet containing a weekly increasing concentrate portion of 20%, 40%, 60%, and 80% over 28d. At d 7, 14, 21, and 28, one group (named C20, C40, C60, and C80 according to the last concentrate level that they received) was killed and the ruminal microbiome was collected. Ruminal acidosis was not detected in any of the goats during the experiment. Nonetheless, ruminal pH dropped sharply from 6.2 to 5.7 (P<0.05) when dietary concentrate increased from 40% to 60%. A combined metagenome and metatranscriptome sequencing approach identified that this was linked to a sharp decrease in the abundance and expression of genes encoding nicotinamide adenine dinucleotide (NAD)-dependent lactate dehydrogenase (nLDH), catalyzing the enzymatic conversion of pyruvate to lactate (P<0.01), whereas the expression of two genes encoding NAD-independent lactate dehydrogenase (iLDH), catalyzing lactate oxidation to pyruvate, showed no significant concomitant change. Abundance and expression alterations for nLDH- and iLDH-encoding genes were attributable to bacteria from Clostridiales and Bacteroidales, respectively. By analyzing the gene profiles of 9 metagenome bins (MAG) with nLDH-encoding genes and 5 MAG with iLDH-encoding genes, we identified primary and secondary active transporters as being the major types of sugar transporter for lactate-producing bacteria (LPB) and lactate-utilizing bacteria (LUB), respectively. Furthermore, more adenosine triphosphate was required for the phosphorylation of sugars to initiate their catabolic pathways in LPB compared to LUB. Thus, the low dependence of sugar transport systems and catabolic pathways on primary energy sources supports the acid tolerance of LUB from Bacteroidales. It favors ruminal lactate utilization during the adaptation of goats to a high-concentrate diet. This finding has valuable implications for the development of measures to prevent RA.

ABC Transporters in Bacterial Nanomachineries.

Members of the superfamily of ABC transporters are found in all domains of life. Most of these primary active transporters act as isolated entities and export or import their substrates in an ATP-dependent manner across biological membranes. However, some ABC transporters are also part of larger protein complexes, so-called nanomachineries that catalyze the vectorial transport of their substrates. Here, we will focus on four bacterial examples of such nanomachineries: the Mac system providing drug resistance, the Lpt system catalyzing vectorial LPS transport, the Mla system responsible for phospholipid transport, and the Lol system, which is required for lipoprotein transport to the outer membrane of Gram-negative bacteria. For all four systems, we tried to summarize the existing data and provide a structure-function analysis highlighting the mechanistical aspect of the coupling of ATP hydrolysis to substrate translocation.

Mechanisms of regulation of motility of the gastrointestinal tract and the hepatobiliary system under the chronic action of nanocolloids

Modern cutting edge technologies of chemical synthesis enable the production of unique nanostructures with excess energy and high reactivity. Uncontrolled use of such materials in the food industry and pharmacology entail a risk for the development of a nanotoxicity crisis. Using the methods of tensometry, mechanokinetic analysis, biochemical methods, and bioinformatics, the current study showed that chronic (for six months) intragastrical burdening of rats with aqueous nanocolloids (AN) ZnO and TiO2 caused violations of the pacemaker-dependent mechanisms of regulation of spontaneous and neurotransmitter-induced contractions of the gastrointestinal tract (GIT) smooth muscles (SMs), and transformed the contraction efficiency indices (AU, in Alexandria units). Under the same conditions, the fundamental principle of distribution of physiologically relevant differences in the numeric values of the mechanokinetic parameters of spontaneous SM contractions between different parts of GIT is violated, which can potentially cause its pathological changes. Using molecular docking, typical bonds in the interfaces of the interaction of these nanomaterials with myosin II, a component of the contractile apparatus of smooth muscle cells (SMC) were investigated. In this connection, the study addressed the question of possible competitive relations between ZnO and TiO2 nanoparticles and actin molecules for binding sites on the myosin II actin-interaction interface. In addition, using biochemical methods, it was shown that chronic long-term exposure to nanocolloids causes changes in the primary active ion transport systems of cell plasma membranes, the activity of marker liver enzymes and disrupts the blood plasma lipid profile, which indicates the hepatotoxic effect of these nanocolloids.

The Switch and Reciprocating Models for the Function of ABC Multidrug Exporters: Perspectives on Recent Research.

ATP-binding cassette (ABC) transporters comprise a large superfamily of primary active transporters, which are integral membrane proteins that couple energy to the uphill vectorial transport of substrates across cellular membranes, with concomitant hydrolysis of ATP. ABC transporters are found in all living organisms, coordinating mostly import in prokaryotes and export in eukaryotes. Unlike the highly conserved nucleotide binding domains (NBDs), sequence conservation in the transmembrane domains (TMDs) is low, with their divergent nature likely reflecting a need to accommodate a wide range of substrate types in terms of mass and polarity. An explosion in high resolution structural analysis over the past decade and a half has produced a wealth of structural information for ABCs. Based on the structures, a general mechanism for ABC transporters has been proposed, known as the Switch or Alternating Access Model, which holds that the NBDs are widely separated, with the TMDs and NBDs together forming an intracellular-facing inverted "V" shape. Binding of two ATPs and the substrate to the inward-facing conformation induces a transition to an outward conformation. Despite this apparent progress, certainty around the transport mechanism for any given ABC remains elusive. How substrate binding and transport is coupled to ATP binding and hydrolysis is not known, and there is a large body of biochemical and biophysical data that is at odds with the widely separated NBDs being a functional physiological state. An alternative Constant Contact model has been proposed in which the two NBSs operate 180 degrees out of phase with respect to ATP hydrolysis, with the NBDs remaining in close proximity throughout the transport cycle and operating in an asymmetric allosteric manner. The two models are discussed in the light of recent nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses of three ABC exporters.

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.

From your kidneys to your eyes: lessons from computational kidney models

Purpose: The purpose of this review is to describe computational models that have been developed for studying kidney function and howthese models may be adapted to study the eyes. Methods: We derive equations for modeling solute andwater transport across epithelial cell membranes in the kidney. These equations describe mass conservation, as well membrane transport via cotransporters, exchangers, and primary active transport. Results: Wedescribe howcomputational models of renal transport have been applied to investigate kidney function in physiological and pathophysiological conditions. Conclusion: The computational models herein described for the kidney may be adapted to study ocular functions and dysfunction.

Structure and function of H+/K+ pump mutants reveal Na+/K+ pump mechanisms

Ion-transport mechanisms evolve by changing ion-selectivity, such as switching from Na+ to H+ selectivity in secondary-active transporters or P-type-ATPases. Here we study primary-active transport via P-type ATPases using functional and structural analyses to demonstrate that four simultaneous residue substitutions transform the non-gastric H+/K+ pump, a strict H+-dependent electroneutral P-type ATPase, into a bona fide Na+-dependent electrogenic Na+/K+ pump. Conversion of a H+-dependent primary-active transporter into a Na+-dependent one provides a prototype for similar studies of ion-transport proteins. Moreover, we solve the structures of the wild-type non-gastric H+/K+ pump, a suitable drug target to treat cystic fibrosis, and of its Na+/K+ pump-mimicking mutant in two major conformations, providing insight on how Na+ binding drives a concerted mechanism leading to Na+/K+ pump phosphorylation.

Plastic structures for diverse substrates: A revisit of human ABC transporters

ATP-binding cassette (ABC) superfamily is one of the largest groups of primary active transporters that could be found in all kingdoms of life from bacteria to humans. In humans, ABC transporters can selectively transport a wide spectrum of substrates across membranes, thus playing a pivotal role in multiple physiological processes. In addition, due to the ability of exporting clinic therapeutics, some ABC transporters were originally termed multidrug resistance proteins. Increasing investigations of human ABC transporters in recent years have provided abundant information for elucidating their structural features, based on the structures at distinct states in a transport cycle. This review focuses on the recent progress in human ABC structural analyses, substrate binding specificities, and translocation mechanisms. We dedicate to summarize the common features of human ABC transporters in different subfamilies, and to discuss the possibility to apply the fast-developing techniques, such as cryogenic electron microscopy, and artificial intelligence-assisted structure prediction, for future studies.

Use of a Short, In‐Class, Free Response Activity to Assess Student Understanding of Core Concepts of the Cell Membrane in an Undergraduate Physiology Course

Students in an upper division, stand‐alone animal physiology course at Missouri Western State University are required to have completed a freshman‐level introductory cell biology course and a sophomore‐level genetics course as prerequisites. These prerequisite courses include discussion of the eukaryotic cell membrane and basic properties and functions of the membrane. However, many students remember the basic information about membrane structure but have difficulty relating that structure to membrane function and related topics in our physiology course, including vesicular transport, primary and secondary active transport, osmosis, and current flow across the membrane. To gain a better understanding of what students recall about the cell membrane before covering membrane structure and function in class, students were given an open‐ended prompt, “What do you know about the structure and function of the animal cell membrane?” The students were allowed to work in small groups or alone, and could generate their response as drawings, written statements, or both. They were given 10 minutes to construct a response. These student responses were analyzed using the conceptual framework for the “cell membrane” core concept recently published by Michael and Modell (Adv Physiol Educ 2019). This activity was repeated 2 weeks after finishing discussing of the cell membrane. Prior to covering this content in class, 100% (19 out of 19) submissions included a drawing or description of the cell membrane as a phospholipid bilayer, and 84% of submissions included integral and peripheral membrane proteins. Similar percentages of students included these concepts in the post‐coverage assessment. However, other components of the “cell membrane” conceptual framework were included much less frequently or not at all prior to covering the content in class, but improved dramatically afterwards. In the pre‐assessment, only 11% of responses included solute concentration gradients or selective permeability, while 70% of responses in the post‐assessment included this concepts. In the pre‐assessment less than 10% mentioned vesicular transport, receptors, or channel proteins; after covering the content in class, these topics were included by 15%, 45%, and 35% of student responses, respectively. This quick activity provides valuable information about what students recall from prior coursework and what concepts need to be revisited before moving into more detailed discussion of cell membrane function. When used in addition to objective exam or assignment questions, this free‐response activity can also provide a complementary view of student understanding of core concepts of the cell membrane.

6-Amino-2-(4-fluorophenyl)-4-(trifluoromethyl)quinoline: Insight into the Crystal Structure, Hirshfeld Surface Analysis and Computational Study

In this work we report detailed structural and computational studies of 6-amino-2-(4-fluorophenyl)-4-(trifluoromethyl)quinoline (1). The structure is stabilized by the intramolecular N–H···N hydrogen bonds, C–H···F interactions, π···π interactions and C–F···F–C interactions. The listed interactions are also reflected on the Hirshfeld surfaces as well as the corresponding 2D fingerprint plots, which revealed that the crystal packing of 1 is mainly dictated by highly favored H···H and H···F contacts followed by also favored H···N, C···C and F···F contacts. The DFT calculations were performed to verify the structure of 1 as well as its electronic and optical properties. Compound 1 was predicted to exhibit preferred results for drug candidates in five parameters, namely lipophilicity, size, polarity, insolubility and flexibility. Furthermore, it was predicted that 1 is likely a potential inhibitor of family A G protein-coupled receptor and kinase; electrochemical transporter and voltage-gated ion channel; oxidoreductase, hydrolase, protease, family C G protein-coupled receptor and primary active transporter with the probabilities of 20.0%, 13.3% and 6.7%, respectively. At the same time, 1 was found to be active against Aryl hydrocarbon Receptor (AhR) and Mitochondrial Membrane Potential (MMP), hepatotoxic and mutagenic. According to the BOILED-Egg method for 1 the human blood-brain barrier (BBB) penetration property is negative and gastrointestinal absorption property is positive with the positive PGP effect on the molecule.

Regulation of sperm hyperactivation by transporters involved in Na+ homeostasis

Mammalian sperm have to undergo several physiological and biochemical changes to be fertilization-competent. These changes are collectively called "capacitation". Capacitated sperm show frantic whiplash-like flagellar movement called "hyperactivation". Recently, it has been reported that treatments of sperm to enhance hyperactivation improve in vitro fertilization (IVF) and development of the fertilized embryos. This fact indicates that hyperactivation is an attractive target to improve assisted reproductive technologies. However, the detailed mechanisms which regulate hyperactivation have not been fully elucidated. Recently, it was found out that Na+ and K+ concentration of the oviductal fluid considerably differs from those in the medium used for IVF. Thus, the effect of the Na+ and K+ concentration on sperm hyperactivation was investigated using hamsters as a model. The results revealed that hamster sperm hyperactivation was suppressed by extracellular Na+ via an action of Na+/Ca2+ exchanger. Moreover, it was shown that the activity of testis specific isoform of Na+/K+ ATPase (NKA) α subunit, α4, is necessary for the hyperactivation-associated change of the flagellar movement. By contrast, there was no significant effect of raising the K+ concentration up to 20 mM on sperm hyperactivation although it significantly depolarized the membrane potential. These results suggests that transporters involved in primary and secondary active transport which regulates cellular Na+ homeostasis has a crucial role in the regulation of hyperactivation, and these transporters can be an attractive target for the improvement of assisted reproductive technologies.

Structural and thermodynamic insights into a novel Mg2+-citrate-binding protein from the ABC transporter superfamily.

More than one third of proteins require metal ions to accomplish their functions, making them obligatory for the growth and survival of microorganisms in varying environmental niches. In prokaryotes, besides their involvement in various cellular and physiological processes, metal ions stimulate the uptake of citrate molecules. Citrate is a source of carbon and energy and is reported to be transported by secondary transporters. In Gram-positive bacteria, citrate molecules are transported in complex with divalent metal ions, whereas in Gram-negative bacteria they are translocated by Na+/citrate symporters. In this study, the presence of a novel divalent-metal-ion-complexed citrate-uptake system that belongs to the primary active ABC transporter superfamily is reported. For uptake, the metal-ion-complexed citrate molecules are sequestered by substrate-binding proteins (SBPs) and transferred to transmembrane domains for their transport. This study reports crystal structures of an Mg2+-citrate-binding protein (MctA) from the Gram-negative thermophilic bacterium Thermus thermophilus HB8 in both apo and holo forms in the resolution range 1.63-2.50 Å. Despite binding various divalent metal ions, MctA possesses the coordination geometry to bind its physiological metal ion, Mg2+. The results also suggest an extended subclassification of cluster D SBPs, which are known to bind and transport divalent-metal-ion-complexed citrate molecules. Comparative assessment of the open and closed conformations of the wild-type and mutant MctA proteins suggests a gating mechanism of ligand entry following an `asymmetric domain movement' of the N-terminal domain for substrate binding.

Comparative Analyses of the Transport Proteins Encoded within the Genomes of nine Bifidobacterium Species

The human microbiome influences human health in both negative and positive ways. Studies on the transportomes of these organisms yield information that may be utilized for various purposes, including the identification of novel drug targets and the manufacture of improved probiotic strains. Moreover, these genomic analyses help to improve our understanding of the physiology and metabolic capabilities of these organisms. The present study is a continuation of our studies on the transport proteins of the major gut microbes. Bifidobacterium species are essential members of the human gut microbiome, and they initiate colonization of the gut at birth, providing health benefits that last a lifetime. In this study we analyze the transportomes of nine bifidobacterial species: B. adolescentis, B. animalis, B. bifidum, B. breve, B. catenulatum, B. dentium, B. longum subsp. infantis, B. longum subsp. longum, and B. pseudocatenulatum. All of these species have proven probiotic characteristics and exert beneficial effects on human health. Surprisingly, we found that all nine of these species have similar pore-forming toxins and drug exporters that may play roles in pathogenesis. These species have transporters for amino acids, carbohydrates, and proteins, essential for their organismal lifestyles and adaption to their respective ecological niches. The strictly probiotic species, B. bifidum, however, contains fewer such transporters, thus indicative of limited interactions with host cells and other gut microbial counterparts. The results of this study were compared with those of our previous studies on the transportomes of multiple species of Bacteroides, Escherichia coli/Salmonella, and Lactobacillus. Overall, bifidobacteria have larger transportomes (based on percentages of total proteins) than the previously examined groups of bacterial species, with a preference for primary active transport systems over secondary carriers. Taken together, these results provide useful information about the physiologies and pathogenic potentials of these probiotic organisms as reflected by their transportomes.

Mechanistic Dissection of Cu(I)‐translocating P‐type ATPase Pumps by I n‐vitro Real‐Time Transport in Artificial Lipid Bilayer Vesicles

Copper is an essential element in biological systems where it plays key roles as a co-factor in diverse cuproenzymes and in signaling processes. Cu(I) transporting P-type ATPases (CopA) are primary active transmembrane transporter proteins that utilize the energy of ATP hydrolysis to catalyze the extrusion of Cu(I) ions across cell membranes. Their activity plays a gatekeeper role in controlling intracellular Cu(I) levels and homeostasis, a key process for survival in all kingdoms of life. In humans, mutations in these Cu(I) pumps lead to the Menkes syndrome and the Wilson's disease. In pathogenic bacteria CopA proteins can act as virulence factors to overcome the Cu(I) stress generated by the host defense system, thereby representing a potential therapeutic target against pathogens causing various fatal diseases. Even though the structure and function of these transporters has been established, the overall mechanism for substrate translocation, their transport kinetic parameters and electrogenicity remain elusive. In this work we developed a novel coupled experimental platform, centered on multiple fluorescence reporter probes that permits to study substrate, the transport mechanism and translocation kinetics in real-time in Cu(I)-pumps reconstituted in a native-like environment. We reconstituted the model Cu(I)-pump from E. coli (EcCopA) and its inactive mutants in artificial small unilamellar vesicles (proteoliposomes). We quantitatively characterized the chemistry underlying substrate transport, putative counterions and charge translocation by encapsulating in the liposome lumen fluorescence detector probes (CTAP-3, Pyranine and Oxonol VI) responsive to diverse stimuli (Cu(I) substrate, pH and transmembrane potential). The study established that Cu(I) pumps are primary-active uniporters and electrogenic pumps that build a transmembrane potential by translocating one positive charge (Cu(I)) per ATP hydrolysis cycle without coupling it to counterion transport. This analysis further reveals mechanistic differences in substrate translocation by CopA proteins in comparison to other characterized P-type ATPase classes. Moreover, the developed approach can be extended as a general biophysical tool to investigate other P-type ATPase pumps in which the mechanism of substrate translocation has not yet been established.

De novo macrocyclic peptides dissect energy coupling of a heterodimeric ABC transporter by multimode allosteric inhibition.

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters involved in a multitude of physiological processes and human diseases. Despite considerable efforts, it remains unclear how ABC transporters harness the chemical energy of ATP to drive substrate transport across cell membranes. Here, by random nonstandard peptide integrated discovery (RaPID), we leveraged combinatorial macrocyclic peptides that target a heterodimeric ABC transport complex and explore fundamental principles of the substrate translocation cycle. High-affinity peptidic macrocycles bind conformationally selective and display potent multimode inhibitory effects. The macrocycles block the transporter either before or after unidirectional substrate export along a single conformational switch induced by ATP binding. Our study reveals mechanistic principles of ATP binding, conformational switching, and energy transduction for substrate transport of ABC export systems. We highlight the potential of de novo macrocycles as effective inhibitors for membrane proteins implicated in multidrug resistance, providing avenues for the next generation of pharmaceuticals.

Role of Transportome in the Gills of Chinese Mitten Crabs in Response to Salinity Change: A Meta-Analysis of RNA-Seq Datasets.

Chinese mitten crab (CMC) or Eriocheir sinensis is a strong osmoregulator that can keep rigorous cellular homeostasis. CMC can flourish in freshwater, as well as seawater, habitats and represents the most important species for freshwater aquaculture. Salt stress can have direct effects on several stages (e.g., reproduction, molting, growth, etc.) of the CMC life cycle. To get a better overview of the genes involved in the gills of CMC under different salinity conditions, we conducted an RNA-Seq meta-analysis on the transcriptomes of four publicly available datasets. The meta-analysis identified 405 differentially expressed transcripts (DETs), of which 40% were classified into various transporter classes, including accessory factors and primary active transporters as the major transport classes. A network analysis of the DETs revealed that adaptation to salinity is a highly regulated mechanism in which different functional modules play essential roles. To the best of our knowledge, this study is the first to conduct a transcriptome meta-analysis of gills from crab RNA-Seq datasets under salinity. Additionally, this study is also the first to focus on the differential expression of diverse transporters and channels (transportome) in CMC. Our meta-analysis opens new avenues for a better understanding of the osmoregulation mechanism and the selection of potential transporters associated with salinity change.

Structure and Function of Hepatobiliary ATP Binding Cassette Transporters.

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

Structural insights into sodium transport by the oxaloacetate decarboxylase sodium pump.

The oxaloacetate decarboxylase sodium pump (OAD) is a unique primary-active transporter that utilizes the free energy derived from oxaloacetate decarboxylation for sodium transport across the cell membrane. It is composed of 3 subunits: the α subunit catalyzes carboxyl-transfer from oxaloacetate to biotin, the membrane integrated β subunit catalyzes the subsequent carboxyl-biotin decarboxylation and the coupled sodium transport, the γ subunit interacts with the α and β subunits and stabilizes the OAD complex. We present here structure of the Salmonella typhimurium OAD βγ sub-complex. The structure revealed that the β and γ subunits form a β3γ3 hetero-hexamer with extensive interactions between the subunits and shed light on the OAD holo-enzyme assembly. Structure-guided functional studies provided insights into the sodium binding sites in the β subunit and the coupling between carboxyl-biotin decarboxylation and sodium transport by the OAD β subunit.

A single power stroke by ATP binding drives substrate translocation in a heterodimeric ABC transporter.

ATP-binding cassette (ABC) transporters constitute the largest family of primary active transporters, responsible for many physiological processes and human maladies. However, the mechanism how chemical energy of ATP facilitates translocation of chemically diverse compounds across membranes is poorly understood. Here, we advance the quantitative mechanistic understanding of the heterodimeric ABC transporter TmrAB, a functional homolog of the transporter associated with antigen processing (TAP) by single-turnover analyses at single-liposome resolution. We reveal that a single conformational switch by ATP binding drives unidirectional substrate translocation. After this power stroke, ATP hydrolysis and phosphate release launch the return to the resting state, which facilitates nucleotide exchange and a new round of substrate binding and translocation. In contrast to hitherto existing steady-state assays, our single-turnover approach uncovers the power stroke in substrate translocation and the tight chemomechanical coupling in these molecular machines.