Exploring the role of residues in the intracellular loop of hZIP4 in the binding of Zn

Zinc is an essential micronutrient. It plays a role in stabilizing transcription factors in order to maintain normal cellular function. When the concentration of zinc is not regulated within cells, pathological conditions such as cancers, gastrointestinal tract and skin disorders develop. Although dysregulation of cellular zinc results in several pathological conditions, the mechanism of zinc transport is not wellunderstood. Elucidating this mechanism may provide insight into treating diseases associated with improper zinc regulation. Human ZIP4 is a member of the ZIP family of proteins that holds a role in the intestinal absorption of zinc by increasing the cytosolic concentration of zinc inside of cells lining the gastrointestinal tract. When this protein is mutated, the disease acrodermatitis enteropathica (AE) can develop. In this project, the role of the intracellular loop between transmembrane domains 3 and 4 of hZIP4, M3M4, is explored in zinc binding. A fluorescence based assay is used to measure the zinc binding affinity of M3M4 to zinc following covalent labeling of M3M4 with diethylpyrocarbonate, maleimide, or N,-N’ dicyclohexylcarbodiimide. The binding affinity to zinc to targeted mutant constructs is examined and the structural changes of M3M4 in the presence and absence of zinc are explored. The results of these experiments reveal that M3M4 binds zinc with high affinity and histidine residues are responsible for zinc coordination. Binding of zinc to M3M4 is shown to result in a conformational change that may have a regulatory effect on zinc transport.