A homology-based 3D model and structure–function studies reveal key elements for divalent metal ion transporter ZIP8 (SLC39A8) function

The divalent metal transporter ZIP8 (Zrt/Irt-like protein 8; SLC39A8) plays a pivotal role in maintaining the homeostasis of essential micronutrients, such as manganese (Mn²⁺), zinc (Zn²⁺), and iron (Fe²⁺). Genetic variants of SLC39A8 have been associated with a variety of human diseases, including neuropsychiatric disorders, Crohn's disease, and obesity. To gain insight into ZIP8-mediated metal transport, we generated a homology-based 3D model and identified the amino acid residues constituting metal-binding sites 1 (M1) and 2 (M2). Mutagenesis of residues N315, E344, and D318, which form M2, resulted in a complete loss of function, suggesting that M2 plays a central role in the binuclear metal center of ZIP8. Conversely, mutagenesis of residues H314, E343, and D410, which form M1, retained functional activity but with significant alterations: Residue H314 was found to affect substrate selectivity, whereas residues E344 and D410 were identified as essential for the transport of Fe²⁺ and Mn²⁺. Furthermore, residue H347 was found to influence the metal transport turnover rates. These findings indicate that M1 provides accessory functions to ZIP8 activity, including maximal transport rates and/or enhanced substrate selectivity. Furthermore, the present study provides the first direct evidence for Zn²⁺-HCO₃^- cotransport by human ZIP8 and provides insights into HCO₃^- modulation of metal transport. In addition, we have identified a novel metal-binding site, termed M4, formed by residues D311, E348, and D351. Overall, the present study reveals new insights into the structure and metal transport function of ZIP8 and provides a new framework for interpreting functional defects and designing potential therapeutic interventions.