Metals such as iron and manganese are essential to many physiological processes including oxygen transport and energy metabolism. But overabundance of these metals is toxic, and their physiological levels are therefore tightly regulated. Nramps (natural resistance-associated macrophage proteins) are membrane transporters that import divalent metal ions into cells. Nramps are important for both divalent metal uptake from the diet and cellular import of metals into the cytosol. Nramps are therefore critical factors in maintaining homeostasis of divalent metals, particularly iron and manganese. Nramp proteins are conserved from bacteria to man, and in many bacterial species they are the principal manganese import system. Nramps are metal-proton symporters, using a pH gradient to drive the co- transport of divalent metals and protons. The overall goal is to determine the molecular mechanism of metal ion-proton symport by the Nramp family of proteins through biochemical, computational, and structural studies of bacterial Nramp proteins. We recently determined the crystal structure of a bacterial Nramp homolog, which serves as a scaffold for generating hypotheses and interpreting data. We have developed a range of in vivo and in vitro activity assays, which we will use to define how metal selectivity is encoded in the sequences of Nramp family transporters. We will also use these assays to understand how protons and protonation events influence the transport cycle. We will study the conformation changes during metal transport using biochemical approaches, engineer constructs that stabilize particular conformational states for high-resolution structure determination by x-ray crystallography, and molecular dynamic simulations in various protonation states to model dynamics produced by protonation or deprotonation events. Our overall goal is to establish an atomic- resolution model of the proton-coupled metal ion transport cycle. While Nramps are part of the well-characterized LeuT-fold superfamily of transporters, they are unusual because they are neither sodium-coupled symporters nor antiporters. Our proposed research focuses on several of Nramps? unique features and will thus expand our knowledge of the mechanistic diversity enabled by the LeuT fold. Both bacterial and mammalian Nramp proteins have an impact on human health. Bacterial Nramps increase pathogenicity by facilitating the uptake of essential divalent metals. Human Nramps are particularly important in immunity to intracellular pathogens, liver and blood homeostasis, and brain function. Nramps have been implicated in numerous pathologic conditions including autoimmune diseases, anemia and Parkinson disease. The proposed basic research will have a major impact on the field by providing sorely needed mechanistic information on the biomedically relevant class of Nramp transporters. These structural and mechanistic insights into metal ion transport by Nramp proteins can eventually contribute to the development of drugs and therapeutic strategies to treat disorders resulting from imbalances in metal ion homeostasis.

Public Health Relevance

Divalent metal ions like iron and manganese are important for many cellular functions. Iron is particularly important in the blood to help transport oxygen, and in mitochondria that energize cells. Manganese is most important to enzymes that keep neurons healthy in the brain. Many disorders are caused by imbalances in the levels of metals, including anemia and manganism (Parkinson-like disease caused by excess manganese). The goal is to determine how Nramp-family proteins select and import these metal ions into cells and understand at the atomic level how the metal ions are transported across membranes. To this end, we will use a range of biochemical assays along with x-ray crystallography, computer simulations and bioinformatics analyses. These proteins are also involved in the uptake of toxic metals like lead. The knowledge gained by this research will help in the design of drugs and therapies for disorders caused by imbalances in essential and/or toxic metals.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Anderson, Vernon
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Harvard University
Schools of Arts and Sciences
United States
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Nicoludis, John M; Gaudet, Rachelle (2018) Applications of sequence coevolution in membrane protein biochemistry. Biochim Biophys Acta Biomembr 1860:895-908