Structure-function analysis of ferroportin defines the binding site and an alternative mechanism of action of hepcidin

Sharraya Aschemeyer, Bo Qiao, Deborah Stefanova, Erika V. Valore, Albert C. Sek, T. Alex Ruwe, Kyle R. Vieth, Grace Jung, Carla Casu, Stefano Rivella, Mika Jormakka, Bryan Mackenzie, Tomas Ganz and Elizabeta Nemeth

Key points

  • Analysis of mutations causing non-classical ferroportin (Fpn) disease defined the hepcidin-binding site in the central cavity of Fpn.

  • Hepcidin inhibits iron export through Fpn not only by causing Fpn endocytosis but also by occluding the transporter.


Non-classical Ferroportin Disease is a form of hereditary hemochromatosis caused by mutations in the iron transporter ferroportin (Fpn), resulting in parenchymal iron overload. Fpn is regulated by the hormone hepcidin, which induces Fpn endocytosis and cellular iron retention. We characterized 11 clinically-relevant and 5 nonclinical Fpn mutations using stably transfected, inducible, isogenic cell lines. All clinical mutants were functionally resistant to hepcidin as a consequence of either impaired hepcidin binding or impaired hepcidin-dependent ubiquitination despite intact hepcidin binding. Mapping the residues onto two computational models of the human Fpn structure indicated that i) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, ii) hepcidin binding occurred within the central cavity of Fpn, iii) hepcidin interacted with up to 4 helices, and iv) hepcidin binding should occlude Fpn and interfere with iron export independently of endocytosis. We experimentally confirmed hepcidin-mediated occlusion of Fpn in the absence of endocytosis in multiple cellular systems: HEK293 cells expressing an endocytosis-defective Fpn mutant (K8R), Xenopus oocytes expressing WT or K8R Fpn, and mature human RBCs. We conclude that non-classical Ferroportin Disease is caused by Fpn mutations that decrease hepcidin binding or hinder conformational changes required for ubiquitination and endocytosis of Fpn. The newly documented ability of hepcidin and its agonists to occlude iron transport may facilitate the development of broadly effective treatments for hereditary iron overload disorders.

  • Submitted May 23, 2017.
  • Accepted November 30, 2017.