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1.
FEBS Open Bio ; 11(1): 26-34, 2021 01.
Article in English | MEDLINE | ID: mdl-33190422

ABSTRACT

Ferroportin (Fpn) is an essential mammalian iron transporter that is negatively regulated by the hormone hepcidin. Our current molecular understanding of Fpn-mediated iron efflux and regulation is limited due to a lack of biochemical, biophysical and high-resolution structural studies. A critical step towards understanding the transport mechanism of Fpn is to obtain sufficient quantities of pure and stable protein for downstream studies. As such, we detail here an expression and purification protocol for mouse Fpn yielding milligram quantities of pure protein. We have generated deletion constructs exhibiting enhanced thermal stability and which retained iron-transport activity and hepcidin responsiveness, providing a platform for further biophysical studies of Fpn.


Subject(s)
Cation Transport Proteins/isolation & purification , Animals , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Hepcidins/metabolism , Hot Temperature/adverse effects , Mice , Protein Stability , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism
2.
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
3.
PLoS One ; 12(9): e0184366, 2017.
Article in English | MEDLINE | ID: mdl-28880952

ABSTRACT

Hephaestin is a large membrane-anchored multicopper ferroxidase involved in mammalian iron metabolism. Newly absorbed dietary iron is exported across the enterocyte basolateral membrane by the ferrous iron transporter ferroportin, but hephaestin increases the efficiency of this process by oxidizing the transported iron to its ferric form and promoting its release from ferroportin. Deletion or mutation of the hephaestin gene leads to systemic anemia with iron accumulation in the intestinal epithelium. The crystal structure of human ceruloplasmin, another multicopper ferroxidase with 50% sequence identity to hephaestin, has provided a framework for comparative analysis and modelling. However, detailed structural information for hephaestin is still absent, leaving questions relating to metal coordination and binding sites unanswered. To obtain structural information for hephaestin, a reliable protocol for large-scale purification is required. Here, we present an expression and purification protocol of soluble mouse hephaestin, yielding milligram amounts of enzymatically active, purified protein using the baculovirus/insect cell system.


Subject(s)
Membrane Proteins/metabolism , Animals , Binding Sites , Blotting, Western , Cation Transport Proteins/chemistry , Cation Transport Proteins/genetics , Cation Transport Proteins/metabolism , Cell Line , Ceruloplasmin/metabolism , Humans , Kinetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Mutation , Protein Structure, Secondary
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