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1.
Nat Commun ; 9(1): 3075, 2018 08 06.
Article in English | MEDLINE | ID: mdl-30082682

ABSTRACT

Ferroportin (Fpn)-the only known cellular iron exporter-transports dietary and recycled iron into the blood plasma, and transfers iron across the placenta. Despite its central role in iron metabolism, our molecular understanding of Fpn-mediated iron efflux remains incomplete. Here, we report that Ca2+ is required for human Fpn transport activity. Whereas iron efflux is stimulated by extracellular Ca2+ in the physiological range, Ca2+ is not transported. We determine the crystal structure of a Ca2+-bound BbFpn, a prokaryotic orthologue, and find that Ca2+ is a cofactor that facilitates a conformational change critical to the transport cycle. We also identify a substrate pocket accommodating a divalent transition metal complexed with a chelator. These findings support a model of iron export by Fpn and suggest a link between plasma calcium and iron homeostasis.


Subject(s)
Calcium/chemistry , Cation Transport Proteins/chemistry , Animals , Binding Sites , Chelating Agents/chemistry , Crystallography, X-Ray , HEK293 Cells , Homeostasis , Humans , Iron/chemistry , Metals/chemistry , Mutagenesis , Oocytes/metabolism , Protein Conformation , Xenopus
2.
Blood ; 131(8): 899-910, 2018 02 22.
Article in English | MEDLINE | ID: mdl-29237594

ABSTRACT

Nonclassical ferroportin disease (FD) 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 2 computational models of the human Fpn structure indicated that (1) mutations that caused ubiquitination-resistance were positioned at helix-helix interfaces, likely preventing the hepcidin-induced conformational change, (2) hepcidin binding occurred within the central cavity of Fpn, (3) hepcidin interacted with up to 4 helices, and (4) 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 wild-type or K8R Fpn, and mature human red blood cells. We conclude that nonclassical FD 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.


Subject(s)
Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Drug Resistance , Hepcidins/metabolism , Iron/metabolism , Animals , Binding Sites , Cation Transport Proteins/genetics , Cells, Cultured , Computer Simulation , Endocytosis , HEK293 Cells , Hepcidins/agonists , Humans , Mice , Mice, Inbred C57BL , Mutagenesis, Site-Directed , Mutation , Oocytes/cytology , Oocytes/metabolism , Protein Binding , Protein Conformation , Protein Domains , Structure-Activity Relationship , Ubiquitination , Xenopus laevis
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