PROJECT SUMMARY ABCB10 is a human mitochondrial inner membrane ATP binding cassette (ABC) transporter that uses energy from ATP hydrolysis to export a substrate out of the matrix. This transporter is essential for protection against oxidative stress during erythropoiesis (abcb10 knock-out mice die in uterus due to anemia and oxidative damage) and protecting the heart (ABCB10 protein level is upregulated in human ischemic myocardium). Despite its potential clinical relevance for treating anemia and protecting the heart against oxidation, the identity of ABCB10’s substrate was unknown until our group recently identified biliverdin, a heme degradation product with antioxidant properties, as the physiological substrate for this transporter. We have also found that zinc mesoporphyrin, a heme analog, increases the basal ATPase activity of the transporter like substrates do. Identification of these substrates has opened the door to the biochemical and structural studies proposed in this project, which will contribute to a better understanding of the molecular mechanisms by which this important transporter works. Our experimental approach involves the use of functional (ATPase assays), spectroscopic (Luminescence Resonance Energy Transfer, LRET), and mutational analysis of ABCB10 reconstituted in lipid nanodiscs. This experimental system has many advantages for the in vitro study of ABC transporters in a “native- like” lipid bilayer and at physiological temperature. We can produce functional human ABCB10 in bacteria, facilitating the production of the numerous mutants needed for this research. Aim 1 will determine the conformational changes that ABCB10 undergoes during its basal ATP hydrolysis cycle and how those molecular movements are modified during activation by substrate. According to our preliminary data, this aim is expected to prove that ABCB10 functions through small conformational changes. If our hypothesis is correct, our findings will challenge the generally accepted idea that all related ABC exporters follow a similar molecular mechanism. Aim 2 will determine substrate-transporter interactions that are critical for ABCB10’s stimulation. We will study the protein’s ATPase activity and associated conformational changes in response to a) variations in the chemical groups of the substrates and b) mutagenesis of residues in a putative substrate binding pocket. Our preliminary results suggest that the substrate’s carboxyl groups and two arginines in the binding pocket are critical for ABCB10’s stimulation. Mutagenesis of these arginines cause constitutive ABCB10 activation (gain-of-function). Here, we expect to gain information about substrate specificity, find putative inhibitors, identify essential residues in the binding pocket, and define conformational changes that accompany alterations in protein’s function. In general, this project will provide molecular information that can validate current structural models in the ABC transporters field a...