Membrane proteins and iron delivery to cells

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Abstract Our long-term objective is to understand the structural basis for the delivery of iron to cells. In particular we will study the structure of the following three proteins: (/) The transferrin (Tf)-transferrin receptor (TfR) complex. We have determined the structure of the Tf-TfR complex using a soluble construct of the TfR ectodomain lacking the stalk region. The resulting structure strongly suggests that the TfR stalk is involved in Tf binding. We will now determine the structure of the complex in the presence of the stalk and perform functional studies to elucidate the effect of the TfR stalk on iron release from the N-terminal lobe of receptor- bound Tf. (//) The divalent metal ion transporter-1 (DMT1). Iron released from the Tf-TfR complex is transported across the endosomal membrane by DMT1, the same protein that mediates iron uptake from the intestinal lumen through the apical surface of duodenal enterocytes. Mutations in DMT1 cause severe hypochromic microcytic anemia and iron overload. We have expressed mg amounts of the DMT1 ortholog from E. co//. We are using this protein to produce two-dimensional (2D) crystals suitable for electron crystallographic structure determination. In parallel, we will attempt to grow three-dimensional (3D) crystals for X-ray crystallographic structure determination and perform structural studies on other bacterial homologs as well as human DMT1. (Hi)Ferroportin. A second iron transporter, ferroportin, exports iron across the basolateral membrane of duodenal enterocytes to the circulation. Mutations in ferroportin cause type IV hemochromatosis, also known as ferroportin disease. We will express human ferroportin for 2D and later for 3D crystallization trials to determine its structure either by electron or X-ray crystallography. We will then decorate ferroportin2D crystals with the peptide hormone hepcidin to elucidate the binding interaction. Relevance Many proteins depend on iron as a co-factor for redox reactions or ligand coordination, making iron an essential element. The facile conversion between ferrous (Fe2+) and ferric iron (Fe3+) poses significant dangers to living cells, however, because it can lead to the formation of hydroxyl radicals, a major source for oxidative damage to proteins, nucleic acids and lipids. Moreover, under physiological conditions ferric iron forms a highly insoluble hydroxide complex, so that despite its abundance, iron is not easily accessible to cells. Toxicity and insolubility have forced the evolution of highly sophisticated machineries for acquiring, storing, and distributing iron. Malfunctioning of these machineries lead either to iron deficiency disorders or iron overload diseases.