Zinc is essential for any living organism and the second most abundant transition metal element in human after iron. Zinc is thought to bind nearly 3000 human proteins. Neither excessive zinc nor zinc deficiency is good for human health, and therefore zinc homeostasis must be regulated in delicate balance. The Zrt-/Irt- like protein (ZIP) family is responsible for zinc influx from either extracellular space or intracellular vesicles into the cytoplasm.Dietary food is the primary zinc supply for humans. ZIP4 is highly expressed on apical surface of gastrointestinal tract, responsible for zinc uptake from diet. ZIP4 is a transporter with a large extracellular domain (ECD). Apo form pZIP4-ECD from Pteropus Alecto (black fruit bat) crystal structure revealed homodimerization and two subdomains. Previous studies have shown ZIP4-ECD is crucial for optimal zinc uptake. However, underlying mechanism has not been clarified. We examined zinc binding to the isolated pZIP4-ECD using a zinc competing fluorescent dye FluoZin-1, and then located the primary zinc-binding sites with a low micromolar affinity within a histidine-rich loop ubiquitously present in ZIP4 proteins through mutagenesis. Zinc binding to this protease-susceptible loop induces local conformational change, whereas global structure remains unchanged. Mutagenesis and functional study on human ZIP4 by using a cell-based zinc uptake assay indicated that the histidine residues within this loop are not involved in preselection of metal substrate but play a role in promoting zinc transport. The results allowed us to propose the roles of the histidine-rich loop in an efficient zinc uptake.Missense mutations of ZIP4 lead to a life-threatening genetic disorder, Acrodermatitis enteropathica (AE), which causes severe zinc deficiency. Seven out of the reported fifteen mutations are located within ZIP4-ECD, but the impacts of these mutations on protein structure and function have not been systematically studied. Here we characterized these AE-causing mutant variants individually both in vivo and in vitro. Immunostaining, functional assay and biophysical studies suggest that AE is likely to be a protein misfolding disease. Due to defects in protein trafficking, the mutant proteins seem to be retained in the ER and cannot be presented at cell surface, which explains total loss of zinc uptake activity. Mutagenesis and biophysical study on pZIP4-ECD by using circular dichroism revealed secondary structure changes and/or reduction in thermal stability caused by these mutations. Overall, this work revealed the molecular basis of ZIP4 dysfunction in AE-carrying patients and is deemed to be helpful for better therapeutics.
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