PROJECT SUMMARY - Mechanism of Divalent Metal Transport by Nramp-Family Transporters Background: Metals like iron and manganese are essential to many physiological processes including oxygen transport and energy metabolism. But excess or deficiency of these ions leads to health issues—including anemia, hemochromatosis and immune or neurological disorders— and their physiological levels are thus tightly regulated. Nramps (natural resistance-associated macrophage proteins) are symporters that import metal ions and protons into cells, and thus are crucial to maintaining transition metal homeostasis. However, the mechanism of coupling between metal ions and protons is unclear. Structures of bacterial Nramps revealed the binding site for the transition metal ion substrate and a proton pathway formed by a polar residue network in the protein scaffold. Furthermore, evidence is emerging that distant Nramp homologs have variations of the metal-binding sequence motifs and transport other metals like Al3+ and Mg2+. Proposed Research: Our goal is two-fold: (i) develop an atomic-level biophysical understanding of the canonical mechanistic features shared by most eukaryotic and bacterial Nramps; and (ii) contrast these features to those in more distant Nramp-like homologs. We combine sequence bioinformatics and other computational approaches with structural and biochemical analyses. In Aim 1, we investigate canonical features of Nramp transporters using a well-established bacterial Nramp model system. We will determine (1a) the metal ion coordination geometry and affinity and selectivity determinants, (1b) whether proton transport is thermodynamically coupled to metal transport, and (1c) how protonation states of the protein alter conformational dynamics. In Aim 2, we investigate divergent Nramp homologs with noncanonical metal-binding and proton-pathway sequence motifs. We examine how these sequence changes affect (2a) proton transport, (2b) metal selectivity, and (2c) metal coordination geometries. Our two aims synergize to provide in- depth biophysical mechanisms and a broad perspective of this important family of transporters. Impact: Both bacterial and mammalian Nramps impact human health. In bacteria, Nramps help commensal microbes acquire essential transition metals and promote colonization by pathogens. Human Nramps are essential for immunity to intracellular pathogens, liver and blood homeostasis, and brain function. This research on metal ion transport by Nramps provides the biochemical and biophysical grounding necessary to explain their essential role in metal homeostasis at the cellular and organismal level. This knowledge could lead to better therapies for metal-related diseases including anemia, hemochromatosis, and many immune and neurological disorders.