ABC transporters: the power to change

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

Structural diversity of ABC transporters

ATP-binding cassette (ABC) transporters form a large superfamily of ATP-dependent protein complexes that mediate transport of a vast array of substrates across membranes. The 14 currently available structures of ABC transporters have greatly advanced insight into the transport mechanism and revealed a tremendous structural diversity. Whereas the domains that hydrolyze ATP are structurally related in all ABC transporters, the membrane-embedded domains, where the substrates are translocated, adopt four different unrelated folds. Here, we review the structural characteristics of ABC transporters and discuss the implications of this structural diversity for mechanistic diversity.</p

Structural and functional diversity calls for a new classification of ABC transporters

Members of the ATP‐binding cassette (ABC) transporter superfamily translocate a broad spectrum of chemically diverse substrates. While their eponymous ATP‐binding cassette in the nucleotide‐binding domains (NBDs) is highly conserved, their transmembrane domains (TMDs) forming the translocation pathway exhibit distinct folds and topologies, suggesting that during evolution the ancient motor domains were combined with different transmembrane mechanical systems to orchestrate a variety of cellular processes. In recent years, it has become increasingly evident that the distinct TMD folds are best suited to categorize the multitude of ABC transporters. We therefore propose a new ABC transporter classification that is based on structural homology in the TMDs

Structures and functions of mitochondrial ABC transporters

A small number of physiologically important ATP-binding cassette (ABC) transporters are found in mitochondria. Most are half transporters of the B group forming homodimers and their topology suggests they function as exporters. The results of mutant studies point towards involvement in iron cofactor biosynthesis. In particular, ABC subfamily B member 7 (ABCB7) and its homologues in yeast and plants are required for iron-sulfur (Fe-S) cluster biosynthesis outside of the mitochondria, whereas ABCB10 is involved in haem biosynthesis. They also play a role in preventing oxidative stress. Mutations in ABCB6 and ABCB7 have been linked to human disease. Recent crystal structures of yeast Atm1 and human ABCB10 have been key to identifying substrate-binding sites and transport mechanisms. Combined with in vitro and in vivo studies, progress is being made to find the physiological substrates of the different mitochondrial ABC transporters

Substrate Specificity in ABC Transporters Using the E. coli Methionine Import System

ATP-binding cassette (ABC) transporters use the energy of ATP to move substrates across membranes against a concentration gradient. The role of ABC transporters is crucial in several essential cellular functions and mutations in ABC transporters in humans have been linked to several conditions, including cystic fibrosis, liver disease, and diabetes. Despite their central roles in homeostasis, the mechanism of ABC transporters remains poorly understood. Our research is focused on studying an ABC importer in E. coli, as a model system, to examine the mechanism of substrate specificity and transport. The bacterial methionine import system consists of a membrane-embedded transporter, MetNI, and a cognate binding protein, MetQ. Studies have been done of MetQ substrate specificity by purifying several variants of MetQ. Characterized by the binding affinities for methionine derivatives via the Isothermal Titration Calorimeter (ITC). Our data confirms that these mutations affect the binding affinities for methionine derivatives. For some mutations it removes the ability to bind to methionine itself. With these binding affinities in hand, further, experiments with the membrane-embedded transporter, MetNI, will be done in order to dissect the mechanism of ABC transporters

Determining the Mechanical Properties of the E. coli Methionine Transporter

Adenosine Triphosphate Binding Cassette (ABC) transporters constitute a superfamily of active transporters embedded in the cellular membrane. They consist of two highly conserved nucleotide-binding subunits which bind and hydrolyze ATP, and two diverse transmembrane subunits which provide a pathway for the substrate to pass through the membrane. ABC transporters serve a broad range of vital functions. Various conditions like cystic fibrosis and Stargardt disease are caused by defunct ABC transporters, and certain medical complications like antibiotic drug resistance are linked to promiscuous ABC transporters. Despite the importance of these transporters in crucial biological processes, the mechanisms of many transporters are yet to be solved. While many universal features of ABC transporters have been identified, the step-by-step process by which individual transporters move the substrate are a mystery. / To further understand the mechanism of ABC transporters, we are studying the E. coli methionine ABC importer MetNI. Because the bacterium needs to vary methionine import based on cellular needs, MetNI ATPase activity and coupled substrate transport must be properly regulated. Our current goal is to understand the mechanistic details of MetNI ATP binding and hydrolysis using a real-time ATPase assay. Here we present our preliminary work on analyzing the kinetics of MetNI ATP usage under varying conditions and with different mutations. This detailed study of MetNI kinetics will ultimately provide insight into the mechanism of methionine import, which may be more broadly applicable to the ABC transporter superfamily

Unidirectional Transport Mechanism in an ATP Dependent Exporter

ATP-binding cassette (ABC) transporters use the energy of ATP binding and hydrolysis to move a large variety of compounds across biological membranes. P-glycoprotein, involved in multidrug resistance, is the most investigated eukaryotic family member. Although a large number of biochemical and structural approaches have provided important information, the conformational dynamics underlying the coupling between ATP binding/hydrolysis and allocrite transport remains elusive. To tackle this issue, we performed molecular dynamic simulations for different nucleotide occupancy states of Sav1866, a prokaryotic P-glycoprotein homologue. The simulations reveal an outward-closed conformation of the transmembrane domain that is stabilized by the binding of two ATP molecules. The hydrolysis of a single ATP leads the X-loop, a key motif of the ATP binding cassette, to interfere with the transmembrane domain and favor its outward-open conformation. Our findings provide a structural basis for the unidirectionality of transport in ABC exporters and suggest a ratio of one ATP hydrolyzed per transport cycle

ABC transporters as multidrug resistance mechanisms and the development of chemosensitizers for their reversal

One of the major problems related with anticancer chemotherapy is resistance against anticancer drugs. The ATP-binding cassette (ABC) transporters are a family of transporter proteins that are responsible for drug resistance and a low bioavailability of drugs by pumping a variety of drugs out cells at the expense of ATP hydrolysis. One strategy for reversal of the resistance of tumor cells expressing ABC transporters is combined use of anticancer drugs with chemosensitizers. In this review, the physiological functions and structures of ABC transporters, and the development of chemosensitizers are described focusing on well-known proteins including P-glycoprotein, multidrug resistance associated protein, and breast cancer resistance protein

Examining the Bacterial Methionine Transporter Utilizing Soluble Lipid Bilayer Systems

ATP-binding cassette (ABC) transporters are ubiquitous across all kingdoms of life. These highly specific pumps translocate substrates across cell membranes through the energy from ATP binding and hydrolysis. A detailed understanding of ABC transporter mechanism could aid in the treatment of a variety of human disorders in which ABC transporters are defective such as cystic fibrosis. While the structural determinations of ABC transporters have provided critical insights, a detailed molecular understanding of how these proteins work has been precluded by difficulties in the functional study of transporters such as unstable “substitute” mimetic environments. To address this issue, we have turned to a system called nanodiscs, which is a discoidal lipid bilayer encircled by a protein helical belt. Unlike liposomes, nanodiscs have been shown to be a viable system for fluorescent spectroscopic analysis. In preparation for, and as subject to, these kind of analyses, this system has been experimentally proven to be durable in a wide range of experimental conditions, including pH, temperature, and salt concentration

Drug transporters: recent advances concerning BCRP and tyrosine kinase inhibitors

Multidrug resistance is often associated with the (over)expression of drug efflux transporters of the ATP-binding cassette (ABC) protein family. This minireview discusses the role of one selected ABC-transporter family member, the breast cancer resistance protein (BCRP/ABCG2), in the (pre)clinical efficacy of novel experimental anticancer drugs, in particular tyrosine kinase inhibitors