Calcium is an essential cofactor for metal efflux by the ferroportin transporter family

Chandrika N Deshpande,Vicky Xin,Bo Qiao,Mika Jormakka,T Alex Ruwe,Bryan Mackenzie,Erika V Valore,Tomas Ganz,Kyle R Vieth,Elizabeta Nemeth,Ali Shawki

doi:10.1038/s41467-018-05446-4

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.

Highlights

Ferroportin (Fpn)—the only known cellular iron exporter—transports dietary and recycled iron into the blood plasma, and transfers iron across the placenta
We demonstrate that human Fpn-mediated 55Fe efflux in RNA-injected Xenopus oocytes is unaffected by Na+ replacement with choline (Fig. 1a)
We have demonstrated that Ca2+ is a required cofactor in Fpnmediated metal efflux, stimulating iron efflux half-maximally at [Ca2+]O = 0.8–0.9 mM

Summary

Introduction

Ferroportin (Fpn)—the only known cellular iron exporter—transports dietary and recycled iron into the blood plasma, and transfers iron across the placenta. The transport cycle is viewed as a series of ligandinduced conformational changes that include open outward and open inward states Both states were structurally determined for BbFpn, revealing a putative substrate-binding site deep in the Nterminal domain[8]; the binding site assignment was provisional, in part due to its unorthodox location. We use biophysical analyses, functional assays, and sitedirected mutagenesis to explore ion coupling and to identify the substrate site in Fpn. We demonstrate that removal of extracellular Ca2+ abolishes Fpn-mediated iron efflux in Xenopus oocyte and human (HEK) expression systems. The structure of the Ca2+-bound BbFpn reveals a putative metal-binding pocket, and mutagenesis of the corresponding site in the human protein changes the metal specificity of Fpn-mediated transport

Methods

Results

Conclusion