Zinc is an essential metal due to its ubiquity in biological processes. Dietary zinc uptake is principally mediated by the intestinal zinc transporter ZIP4. Human mutations impairing intestinal zinc absorption cause a host of zinc deficiency diseases including acrodermatitis enteropathica (AE), a rare autosomal recessive disorder that is lethal without treatment. In its role of zinc uptake, ZIP4 selectively binds Zn(II, and then transports the bound Zn(II) across the membrane barrier. Understanding the molecular basis of ZIP4 transport is indispensable for unraveling the AE pathogenesis. At present, there is an extreme paucity of structural information on any ZIP proteins. This critical knowledge gap hinders a mechanistic understanding of the zinc transport reaction catalyzed by ZIP4 and other ZIP homologs that are ubiquitous and conserved from bacteria to humans. Our long-term goal is to understand the molecular basis for selective binding and transport of Zn(II) in human ZIP4. This proposed study will focus on a ZIP ortholog from Bordetella bronchiseptica, termed ZIPB. ZIPB is the first member of the ZIP family that can be purified and crystallized. The accessibility to direct biochemical and X-ray crystallographic analyses provides a unique opportunity to explore how zinc binding affinity and selectivity are built into protein structures, and how protei dynamics may reshape zinc-binding sites to render mobility.
In Aim -1, we will identify residues directly involved in zinc binding and translocation.
In Aim -2, we will carry out a comparative analysis of ZIPB and human ZIP4 to determine the functional roles of these residues.
In Aim -3, we will determine the crystal structure of ZIPB, which will provide the basis for homolog modeling of human ZIP4. The proposed work is built on a novel radiolytic labeling technique coupled with a proteomic analysis of ZIPB at a single-residue resolution. The crystallization of ZIPB enables de novo structure determination to obtain the first atomic-level structure model for the ZIP protein family. With these enabling technologies and the availability of ZIPB protein crystals, we are now poised to identify critical functional components of ZIPB (Aim-1), to elucidate molecular mechanisms of zinc binding, transport and functional regulation (Aim-2), and determine the ZIPB structure at an atomic resolution (Aim-3).
Zinc is an essential micronutrient for cell growth and a signaling molecule for cell functions. Zinc transporters in the ZIP protein family constitute a major zinc entry route into intestinal epithelial cells. The fundamental knowledge gained through the proposed structure-function studies will facilitate risk assessment of human ZIP mutations and development of novel therapeutic strategies for zinc deficiency diseases.
