Zinc is the second most abundant transition metal in the body. Despite the fact that hundreds of biomolecules require zinc for proper function and/or structure, the mechanism of zinc transport into cells is not well-understood. ZnT and ZIP proteins are members of the SoLute Carrier (SLC) family of membrane transporters and are one of the principal family of proteins involved in regulating intracellular zinc concentration. ZnT (Zinc Transporters) decrease the cytosolic concentration of zinc, while ZIP (for Zinc or Iron regulated Proteins) transporters function to increase the cytosolic zinc concentration.Mutations in one member of the ZIP family of proteins, the human ZIP4 (hZIP4; SLC39A4) protein, can result in the disease acrodermatitis enteropathica (AE). AE is characterized by growth retardation, diarrhea, behavioral disturbances and neurological disorders. While the cellular distribution of hZIP4 protein expression has been elucidated, the cation specificity, kinetic parameters of zinc transport, residues involved in cation translocation and structural details are unresolved questions. To elucidate cation specificity and kinetic parameters of zinc transport by hZIP4, we have established a high signal to noise zinc uptake assay following heterologous expression of hZIP4 in Xenopus laevis oocytes. The results from our experiments have demonstrated that zinc, copper (II) and nickel can be transported by hZIP4 when the cation concentration is in the micromolar range. We have also identified a nanomolar affinity for copper (II) and zinc transport by hZIP4. In contrast, under nanomolar conditions, nickel can bind, but is not transported by hZIP4. Finally, labeling of hZIP4 with maleimide or DEPC indicates that extracellularly accessible histidine, but not cysteine, residues are required, either directly or indirectly, for cation uptake. The results from our experiments describe at least two coordination sites for divalent cations and provide a new framework to investigate the ZIP family of proteins. Members of the ZIP family of proteins are a central participant in transition metal homeostasis as they function to increase the cytosolic concentration of zinc and/or iron. However the lack of a crystal structure hinders elucidation of the molecular mechanism of ZIP transport proteins. We employed GREMLIN, a co-evolution based contact prediction approach in conjunction with the ROSETTA structure prediction program to construct a structural model of the human (h) ZIP4 transporter. The resulting computational data is best fit by modeling hZIP4 as a dimer. Mutagenesis of residues that comprise a central putative hZIP4 transmembrane transition metal coordination site in the structural model alter the kinetics and specificity of hZIP4. Comparison of the hZIP4 dimer model to all known membrane protein structures identifies the twelve transmembrane monomeric PiPT, a member of the major facilitator superfamily (MFS), as a likely structural homolog. hZIP4 has eight transmembrane domains and encodes a large intracellular loop between transmembrane domains III and IV, M3M4. Previously, it has been postulated that this domain regulates hZIP4 levels in the plasma membrane in a zinc-dependent manner. To elucidate the zinc binding properties of the large intracellular loop of hZIP4, we have recombinantly expressed and purified M3M4 and showed that this fragment binds two zinc ions. Using a combination of site-directed mutagenesis, metal binding affinity assays, and X-ray absorption spectroscopy, we demonstrated that the two Zn2+ ions bind sequentially, with the first Zn2+ binding to a CysHis3 site with a nanomolar binding affinity, and the second Zn2+ binding to a His4 site with a weaker affinity. Circular dichroism spectroscopy revealed that the M3M4 peptide is intrinsically disordered, with only a small structural change induced upon Zn2+ coordination. Our data supports a model in which the intracellular M3M4 domain senses high cytosolic Zn2+ concentrations and regulates the plasma membrane levels of the hZIP4 transporter in response to Zn2+ binding.

Zinc is the second most abundant transition metal in biological systems. This cation is required for the catalytic activity of hundreds of enzymes which mediate protein synthesis, DNA replication and cell division. Despite the central importance of zinc in cellular homeostasis, the mechanism of zinc uptake, compartmentalization and efflux is unknown. Recently, a family of proteins, called ZIP, has been shown to control zinc uptake. Mutations in one of the genes coding for these proteins (ZIP4) can lead to potentially life-threatening diseases like Acrodermatitis Enteropathica and high levels of ZIP4 have been detected in patients suffering from pancreatic cancer. Therefore our goal is to investigate the mechanism of ZIP4 transport and regulation. It was previously shown that the intracellular loop between transmembrane III and IV (M3M4) of ZIP4 is ubiquitinated in the presence of high intracellular zinc which lead to protein degradation. Our initial hypothesis was that the large intracellular domain of ZIP4 (M3M4) is a sensor which detects the intracellular concentration of zinc and regulates the surface expression of ZIP4. In order to test this hypothesis we expressed and purified the M3M4 domain to examine the ability of M3M4 to bind zinc. Our results have demonstrated that M3M4 binds zinc with a 2:1 zinc:protein stoichiometry with nanomolar affinity. We have also shown that upon binding of zinc, M3M4 undergoes a large conformational change.