The N‐terminus of Hepcidin is Essential for Its Interaction with Ferroportin: Structure‐Function Study

Hepcidin is the principal iron‐regulatory hormone. It acts by binding to the iron exporter ferroportin (Fpn), inducing its internalization and degradation, thereby blocking cellular iron efflux. The bioactive hepcidin is a 25 amino acid (aa) peptide with a hairpin structure stabilized by 4 disulfide bonds. We synthesized a series of hepcidin derivatives and determined their bioactivity in a cell line expressing Fpn‐GFP fusion protein, by measuring the degradation of Fpn‐GFP and the accumulation of ferritin after peptide treatment. Bioactivity was also assayed in mice, by measuring hypoferremia after peptide injection. Serial deletion of N‐terminal aa's caused progressive decrease in activity which was completely lost when 5 aa were deleted. Synthetic 3‐aa and 6‐aa N‐terminal peptides alone, however, did not act as agonists or antagonists. Deletion of two C‐terminal aa, or the extension of the N‐terminus by 1 or 5 aa, did not affect peptide activity. Removal of individual disulfide bonds by pairwise C > A substitution did not impact activity in vitro. However, these peptides were less active in vivo, likely due to their decreased stability in circulation. Human G71D or K83R substitutions were fully active in vitro and in vivo. Zebrafish hepcidin, 60% similar to human hepcidin but with conservative substitutions at the N‐terminus, was fully active. Substitution of the 5 N‐terminal aa with the N‐terminus of an unrelated peptide (hbd2), however, abolished the activity. The N‐terminus of hepcidin is essential for the interaction with Fpn but the rest of the structure is permissive for mutations. Supported by NIH DK 065029 and Will Rogers Fund.