Project Summary Copper ions are an essential nutrient for living organisms and are widely used in enzyme-catalyzed redox reactions. However, high levels of copper ions are toxic to cells, including microbial pathogens. Healthcare facilities use copper in touched surfaces to prevent the spread of infectious diseases, and mammalian immune systems can harness the toxicity of copper ions to kill pathogenic bacteria in macrophages. Nevertheless, pathogenic bacteria can resist being killed by copper ions. Therefore, this proposal seeks to understand structure-function relationships for a novel copper ion resistance protein in enterobacteria, a family of Gram-negative bacteria that includes major etiological agents of food-borne illness and hospital-acquired infections worldwide. Our preliminary studies have shown that the lipoprotein DcrB confers resistance to high Cu2+ in Salmonella enterica serovar Typhimurium, a bacterial cause of food-borne illness in humans. The DcrB protein is highly conserved in enterobacteria. We have determined the first three-dimensional structure of a DcrB protein, which revealed it contains the Mog1p/PsbP-like fold, a widespread fold found in proteins that participate in a range of cellular functions. We have identified structural features that are important for the function of DcrB in copper resistance. This proposal seeks to understand how these features of DcrB are essential for its function. Aim 1 focuses on determining how two clusters of residues form an essential functional combination. Aim 2 focuses on investigating how the N-terminal beta-hairpin is essential for the function of DcrB. For both aims, we will use site-directed mutagenesis, genetic, and physiological experiments to probe the structural and biochemical properties for each feature. We will use X-ray crystallography to determine how changes to these features impact the structure of DcrB, and we will use biophysical experiments to investigate the influence of these features on the thermodynamics of folding of DcrB. The proposed research will engage undergraduate students at a primarily undergraduate institution in hands-on biomedical research that will reveal how a novel family of membrane-anchored proteins contributes to copper ion resistance in a medically relevant bacterial species. Results will be applicable to various human pathogens that cause bacterial food-borne illness, plague, and healthcare-associated infections. Knowledge gained from this research will lead to the development of strategies to target bacterial resistance to metal ions and enhance the effective use of copper as an antimicrobial surface. Broadly, this proposal will 1) provide insight into the fundamental biological question of how cells control levels of a toxic metal ion; 2) advance structure-function knowledge for a widespread protein fold; 3) impact human health research by studying this question in bacteria that threaten public health worldwide; and 4) impact scientific ed...