Structural Aspects of Hepcidin-Ferroportin Binding

Hepcidin, a 25-amino acid peptide hormone, is the principal regulator of plasma iron concentrations in a wide range of organisms, from humans to fish. The hepcidin receptor is the iron channel ferroportin (Fpn), which exports iron from duodenal enterocytes, macrophages and hepatocytes into plasma. Hepcidin binding to Fpn results in internalization and degradation of the ligand-receptor complex and reduced iron efflux from cells into plasma. Abnormal production of hepcidin or abnormal interaction with Fpn causes a spectrum of iron disorders. We analyzed the nature of the interaction and critical structural features of hepcidin and Fpn. The binding of hepcidin to Fpn showed an unusual temperature dependence, with loss of binding/internalization at temperatures lower that 15C. To establish whether initiation of internalization stabilized binding, we used Fpn mutated at Tyr302 and Tyr303, which does not internalize and showed that the mutant was still able to bind ¹²⁵I-hepcidin, with a similar EC50 and temperature dependence as wt Fpn. We next addressed Fpn structural features required for interaction with hepcidin. Several Fpn mutations in humans cause a phenotype consistent with resistance to hepcidin. We thus generated Fpn plasmids carrying the specific human mutations, transiently transfected cells with the mutants, and measured ¹²⁵I-hepcidin binding to cells. The results showed that thiol form of Cys326 is essential for hepcidin binding since substitution of this Cys with Ser or Thr preserved the iron exporting function of Fpn but resulted in a complete loss of hepcidin binding, as did the treatment of cells with non-permeable sulfhydryl alkylating agents. The essential role of the C326 residue in hepcidin binding accounts for the early and severe iron overload in patients with C326S or Y substitution. We also showed that the N-terminus of hepcidin is essential for its binding to Fpn. The sequential truncation of five N-terminal residues resulted in a gradual reduction in activity. Ala scanning of the N-terminus showed that Phe4 and Ile6 substitutions resulted in >80% and 50% decrease in binding respectively. To understand the requirements for biological activity at position 4, we tested a series of Phe4 analogues. Substitution with a similar hydrophobic residue, cyclohexylalanine, had no effect on activity; substitution with polar Tyr caused a 50% reduction in activity; substitution with charged residues Lys or Asp resulted in a complete loss of activity, as did substitution with D-Phe. The results indicated that position 4 requires a bulky hydrophobic residue and that the interaction with Fpn is stereospecific. Similarly, substitution of Ile at position 6 with charged residues caused a complete loss of activity. Understanding the molecular framework responsible for hepcidin-Fpn interaction will facilitate the development of drug leads for a range of iron disorders.