Dr. Heather Haemig ‘00
University of Minnesota
Investigation of the metal binding site of MntH, a divalent metal cation/H+ transporter of Escherichia coli
To protect itself, a cell is surrounded by a hydrophobic bilayer membrane. Unfortunately this mode of defense also inhibits the cell from obtaining many essential nutrients and excreting waste products. Therefore, the cell membrane contains numerous proteins that selectively permit molecules to go into and out of the cell across the membrane. Based on genomic sequence data, it is predicted that 30% of the proteins produced by Escherichia coli, Saccharomyces cerevisae, and Homo sapiens are integral membrane proteins. A novel family of membrane proteins has been determined to function as divalent metal ion/hydrogen ion symporters, and homologs of this Natural Resistance-Associated Macrophage protein (Nramp) has been found in plants, animals, and bacteria. The high degree of sequence conservation among family members suggests the structure and function of this family are important to its role in metal ion transport. Forty-two percent of the residues between the human Nramp and MntH are identical allowing information learned from the E. coli transporter to be applied in understanding how the human Nramp transports metal ions across cell membranes. This information can be applied to designing antimicrobial therapies or developing treatment for human diseases like hereditary hemochromatosis which causes an overloading of iron in the body’s tissues and affects 1/400 Caucasians.
Site-directed mutagenesis of E.coli MntH was performed on residues that are highly conserved among the Nramp (Natural Resistance Associated Macrophage Protein) family to which MntH belongs. Negatively charged residues conserved within the family were of particular interest for their possible roles in transport of divalent metal cations across the inner membrane. In addition, transmembrane domain six contains a number of highly conserved residues, which were studied with respect to metal transport. Each mutation to MntH was characterized by transport ability, protein expression, and kinetic properties. The results led to a model for the metal binding site within MntH.