Production of a human mitochondrial ABC transporter in E. coli.

Membrane proteins play important roles in health and disease. Despite their importance, the study of membrane proteins has been significantly limited by the difficulties inherent to their successful expression, purification, and stabilization once they have been extracted from the cell membrane. In addition, expression of human membrane proteins commonly requires the use of expensive and/or time-consuming eukaryotic systems, hence their successful expression in bacteria will be obviously beneficial for experimental research. Furthermore, since lipids can have critical effects on the activity of membrane proteins and given the composition similarities between the inner mitochondrial membrane and the bacterial plasma membrane, production of mitochondrial membrane proteins in E. coli represents a logical choice. Here, we present a novel protocol to produce a human mitochondrial ATP-Binding Cassette (ABC) transporter in E. coli. The function of the three known human mitochondrial ABC transporters is not fully understood, but X-ray crystallography models of ABCB10 produced in insect cells are available. We have successfully expressed and purified ABCB10 from E. coli. The yield is close to that of another bacterial ABC transporter routinely produced in our laboratory under similar conditions. In addition, we can efficiently reconstitute detergent purified ABCB10 into lipid nanodiscs. Measurements of ATPase activity of ABCB10 produced in E. coli show an ATP hydrolysis rate similar to other human ABC transporters. This novel protocol facilitates the production of this human mitochondrial transporter for biochemical, structural, and functional analysis, and can likely be adjusted for production of other mitochondrial transporters.

Stimulation of the human mitochondrial transporter ABCB10 by zinc-mesoporphrin.

Heme biosynthesis occurs through a series of reactions that take place within the cytoplasm and mitochondria, so intermediates need to move across these cellular compartments. However, the specific membrane transport mechanisms involved in the process are not yet identified. The ATP-binding cassette protein ABCB10 is essential for normal heme production, as knocking down this transporter in mice is embryonically lethal and accompanied by severe anemia plus oxidative damage. The role of ABCB10 is unknown, but given its location in the inner mitochondrial membrane, it has been proposed as a candidate to export either an early heme precursor or heme. Alternatively, ABCB10 might transport a molecule important for protection against oxidative damage. To help discern between these possibilities, we decided to study the effect of heme analogs, precursors, and antioxidant peptides on purified human ABCB10. Since substrate binding increases the ATP hydrolysis rate of ABC transporters, we have determined the ability of these molecules to activate purified ABCB10 reconstituted in lipid nanodiscs using ATPase measurements. Under our experimental conditions, we found that the only heme analog increasing ABCB10 ATPase activity was Zinc-mesoporphyrin. This activation of almost seventy percent was specific for ABCB10, as the ATPase activity of a negative control bacterial ABC transporter was not affected. The activation was also observed in cysteine-less ABCB10, suggesting that Zinc-mesoporphyrin's effect did not require binding to typical heme regulatory motifs. Furthermore, our data indicate that ABCB10 was not directly activated by neither the early heme precursor delta-aminolevulinic acid nor glutathione, downsizing their relevance as putative substrates for this transporter. Although additional studies are needed to determine the physiological substrate of ABCB10, our findings reveal Zinc-mesoporphyrin as the first tool compound to directly modulate ABCB10 activity and raise the possibility that some actions of Zinc-mesoporphyrin in cellular and animal studies could be mediated by ABCB10.

Substrate-induced conformational changes in the nucleotide-binding domains of lipid bilayer-associated P-glycoprotein during ATP hydrolysis.

P-glycoprotein (Pgp) is an efflux pump important in multidrug resistance of cancer cells and in determining drug pharmacokinetics. Pgp is a prototype ATP-binding cassette transporter with two nucleotide-binding domains (NBDs) that bind and hydrolyze ATP. Conformational changes at the NBDs (the Pgp engines) lead to changes across Pgp transmembrane domains that result in substrate translocation. According to current alternating access models (substrate-binding pocket accessible only to one side of the membrane at a time), binding of ATP promotes NBD dimerization, resulting in external accessibility of the drug-binding site (outward-facing, closed NBD conformation), and ATP hydrolysis leads to dissociation of the NBDs with the subsequent return of the accessibility of the binding site to the cytoplasmic side (inward-facing, open NBD conformation). However, previous work has not investigated these events under near-physiological conditions in a lipid bilayer and in the presence of transport substrate. Here, we used luminescence resonance energy transfer (LRET) to measure the distances between the two Pgp NBDs. Pgp was labeled with LRET probes, reconstituted in lipid nanodiscs, and the distance between the NBDs was measured at 37 °C. In the presence of verapamil, a substrate that activates ATP hydrolysis, the NBDs of Pgp reconstituted in nanodiscs were never far apart during the hydrolysis cycle, and we never observed the NBD-NBD distances of tens of Å that have previously been reported. However, we found two main conformations that coexist in a dynamic equilibrium under all conditions studied. Our observations highlight the importance of performing studies of efflux pumps under near-physiological conditions, in a lipid bilayer, at 37 °C, and during substrate-stimulated hydrolysis.

Nucleotide dependence of the dimerization of ATP binding cassette nucleotide binding domains.

ATP-binding cassette proteins are ubiquitously present throughout all known genomes. Their basic functional unit possesses two transmembrane domains and two nucleotide-binding domains. The nucleotide-binding domains are responsible for ATP binding and hydrolysis, and their 3-dimensional structure is conserved across ATP-binding cassette proteins. Binding of ATP produces nucleotide-binding domain dimerization, a step necessary for hydrolysis. However, the possibility that nucleotide-binding domains bind and/or hydrolyze nucleotide triphosphates different from ATP has not been explored in detail. Here, we studied that possibility using M. jannaschii MJ0796, a prototypical ATP-binding cassette nucleotide-binding domain. We found that nucleotide-binding domain dimerization occurs as a result of binding to the natural nucleotide triphosphates ATP, GTP, CTP and UTP, and also to the analog ATP-γ-S. All the natural nucleotide triphosphates are hydrolyzed at similar rates, whereas ATP-γ-S is not hydrolyzed. We also found that the non-hydrolyzable ATP analog AMP-PNP, frequently assumed to produce the nucleotide-bound conformation, failed to elicit nucleotide-binding domain dimerization. Our results raise the possibility that not all the nucleotide binding sites of nucleotide-binding domains are occupied by ATP under physiological conditions, and that ATP is not always the nucleotide hydrolyzed to dissociate the nucleotide-binding domain dimers.