Background: ZIP transporters increase the cytosolic concentration of first row transition metals. Results: We have developed a structural model of hZIP4 by combining protein prediction methods with in situ experiments. Conclusion: Analysis of our experiments provides insight into the permeation pathway of hZIP4. Significance: Comparison of this model to membrane transporter crystal structures provides a structural linkage to MFS proteins.

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. The ZIP (Zrt- and Irt-like proteins; SLC39A) family of proteins acts to increase cytosolic concentrations of zinc. 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 and diarrhea, as well as behavioral and neurological disturbances. While the cellular distribution of hZIP4 protein expression has been elucidated, the cation specificity, kinetic parameters of zinc transport, and residues involved in cation translocation are unresolved questions. Therefore, 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 binding affinity where copper(II) and zinc can be transported. In contrast, under these conditions, nickel can bind but is not transported by hZIP4. Finally, labeling of hZIP4 with maleimide or diethylpyrocarbonate indicates that extracellularly accessible histidine, but not cysteine, residues are required, either directly or indirectly, for cation uptake. The results of our experiments identify at least two coordination sites for divalent cations and provide a new framework for investigating the ZIP family of proteins.

Metal ion signaling in biology has been studied extensively with ortho-nitrobenzyl photocages; however, the low quantum yields and other optical properties are not ideal for these applications. We describe the synthesis and characterization of NTAdeCage, the first member in a new class of Zn2+ photocages that utilizes a light-driven decarboxylation reaction in the metal ion release mechanism. NTAdeCage binds Zn2+ with sub-pm affinity using a modified nitrilotriacetate chelator and exhibits an almost 6 order of magnitude decrease in metal binding affinity upon uncaging. In contrast to other metal ion photocages, NTAdeCage and the corresponding Zn2+ complex undergo efficient photolysis with quantum yields approaching 30%. The ability of NTAdeCage to mediate the uptake of 65Zn2+ by Xenopus laevis oocytes expressing hZIP4 demonstrates the viability of this photocaging strategy to execute biological assays.

The human (h) ZIP4 transporter is a plasma membrane protein which functions to increase the cytosolic concentration of zinc. hZIP4 transports zinc into intestinal cells and therefore has a central role in the absorption of dietary zinc. 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. The objective of this research was to examine the zinc binding properties of the large intracellular loop of hZIP4. Therefore, we have recombineantly expressed and purified M3M4 and showed that this domain 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 domain 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.

Metal ion signaling in biology has been studied extensively with ortho-nitrobenzyl photocages; however, the low quantum yields and other optical properties are not ideal for these applications. We describe the synthesis and characterization of NTAdeCage, the first member in a new class of Zn 2+ photocages that utilizes a light-driven decarboxylation reaction in the metal ion release mechanism. NTAdeCage binds Zn 2+ with sub-pM affinity using a modified nitrilotriacetate chelator and exhibits an almost 6 order of magnitude decrease in metal binding affinity upon uncaging. In contrast to other metal ion photocages, NTAdeCage and the corresponding Zn 2+ complex undergo efficient photolysis with quantum yields approaching 30%. The ability of NTAdeCage to mediate the uptake of 65 Zn 2+ by Xenopus laevis oocytes expressing hZIP4 demonstrates the viability of this photocaging strategy to execute biological assays. Keywords cage compounds; coordination compounds; photolysis; X-ray diffraction; zinc The importance of Zn 2+ beyond structural and catalytic functions in proteins has become increasingly apparent. [1] Zinc and iron regulated proteins (ZIPs) increase cytosolic Zn 2+ concentrations; in contrast, zinc transporter (ZnT) proteins decrease cytosolic Zn 2+ . [2,3] Elucidating the function of free or loosely bound Zn 2+ in the cytosol as well as in intracellular compartments remains important; however, the ability to modulate Zn 2+ levels in vivo in a time-resolved manner remains elusive. [4] Free Zn 2+ has been implicated in various signaling pathways, [1] and recently fertilization of oocytes has been shown to trigger "zinc sparks" that serve to initiate meiosis at the beginning stages of embryotic development. [5] The development of new methodologies to simulate fluctuations in Zn 2+ concentrations in a spatiotemporal manner would facilitate the understanding of complex signaling processes.Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201505778. HHS Public Access Author Manuscript Author ManuscriptAuthor Manuscript Author ManuscriptThe photochemistry of ortho-nitrobenzyl (oNB) chromophores initially was recognized as a means to deliver Ca 2+ in a controlled manner to biological receptors using light. [6] We subsequently adapted two Ca 2+ -releasing strategies to develop photocaged complexes for biologically relevant metal ions such as Zn 2+ , Cu + and Fe 3+ . [7,8] In the Cast photocages, decreased electron density on a coordinated aniline nitrogen atom following photolysis lowers chelator binding affinity. [9][10][11] While this strategy can adequately buffer Ca 2+ at typical intracellular resting levels (ca. 100 nM) and simulate biologically relevant concentration increases, [6,12] the reliance on weakly coordinating aniline-based ligands precludes use with Zn 2+ , which experiences tighter intracellular homeostasis. [11] Alternatively, photolytically breaking a carbon-heteroatom bond provides compounds that release metal ions through the reduction ...