essential for by the transporter family.

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 ef fl ux remains incom-plete. Here, we report that Ca 2 + is required for human Fpn transport activity. Whereas iron ef fl ux is stimulated by extracellular Ca 2 + in the physiological range, Ca 2 + is not transported. We determine the crystal structure of a Ca 2 + -bound BbFpn, a prokaryotic orthologue, and fi nd that Ca 2 + is a cofactor that facilitates a conformational change critical to the transport cycle. We also identify a substrate pocket accommodating a divalent transition metal com-plexed with a chelator. These fi ndings support a model of iron export by Fpn and suggest a link between plasma calcium and iron homeostasis. Oocytes in MBM were mounted on the Zeiss LSM 710 META confocal laser-scanning microscope (LSM). GFP fl uorescence was visualized by using the C-45 Apoc- hromat ×10/0.45 W objective, exciting at 488 nm, and detecting fl uorescence in the range 505 – 530 nm with optical slice of ≈ 8µm approximately bisecting the oocyte. 5mM was in the In each experiment, the protein was loaded into the and the solution was loaded in the syringe. The cell was fi lled with the respective degassed buffer. The was injected 25 times ( fi rst injection of 0.2 μ l followed by 24 injections at 1 μ l), with a stirring speed of 750 rpm and 200s intervals between injections. To obtain the effective heat of binding, the heat of dilution, measured by injecting the ligand solution alone into buffer, was subtracted prior to data analysis. The data were fi tted using the ‘ one binding site ’ model using the MicroCal PEAQ-ITC Analysis Software (Malvern Instruments), and the apparent molar reaction entropy ( Δ H °), entropy ( Δ S °), dissociation constant ( K d ), and stoichiometry of binding ( N ) were determined. Our experiments were conducted a minimum of three times to ensure reproducibility. In the fi gures, we show the results of one experiment without statistical analysis. Data availability . Data supporting the fi ndings of this manuscript are available from the corresponding authors upon reasonable request. X-ray structural data and structure factors have been deposited in the Protein Data Bank under accession number 6BTX.