Structural basis of ion transport and inhibition in ferroportin

Abstract

Ferroportin is an iron exporter essential for releasing cellular iron into circulation. Ferroportin is inhibited by a peptide hormone, hepcidin. In humans, mutations in ferroportin lead to ferroportin diseases that are often associated with accumulation of iron in macrophages and symptoms of iron deficiency anemia. Here we present the structures of the ferroportin from the primate Philippine tarsier (TsFpn) in the presence and absence of hepcidin solved by cryo-electron microscopy. TsFpn is composed of two domains resembling a clamshell and the structure defines two metal ion binding sites, one in each domain. Both structures are in an outward-facing conformation, and hepcidin binds between the two domains and reaches one of the ion binding sites. Functional studies show that TsFpn is an electroneutral H+/Fe2+ antiporter so that transport of each Fe2+ is coupled to transport of two H+ in the opposite direction. Perturbing either of the ion binding sites compromises the coupled transport of H+ and Fe2+. These results establish the structural basis of metal ion binding, transport and inhibition in ferroportin and provide a blueprint for targeting ferroportin in pharmacological intervention of ferroportin diseases.

Insights into the Role of the Discontinuous TM7 Helix of Human Ferroportin through the Prism of the Asp325 Residue

“…It is useful here to recall that many MFSs utilize the so-called "proton-motive force" to drive the transport process [28,29], and that there is often a pair of glutamates, aspartates, or histidines, which are able to protonate or deprotonate in the translocation pathway, inside the central cavity of the MFS and in the vicinity of the substrate binding site [28]. This probably includes FPN1 in its IF conformation when the cytoplasmic iron comes to be exported, as very recently suggested by various groups using proteoliposome systems [21,29]. Whether FPN1 is a symporter (Fe 2+ and H + transported in the same direction) or an antiporter (Fe 2+ and H + transported in opposite directions) has not be fully elucidated, but it is important to also remember that cells have a negative resting membrane potential, making export of positively charged Fe 2+ and H + problematic energetically; thus, coupled import of protons is more likely to facilitate iron export.…”

“…A well-studied example is that of Glucose Transporters (GLUTs), which have a broken TM7 helix, with a short TM7b segment playing a key role in extracellular gating, even being directly involved in substrate preference through specific changes in gating dynamics [10,25]. A similar topology is observed for TM7 in the C-lobe of ferroportin [18][19][20][21], although here the exact coupling mechanism between iron binding and extracellular gating is unclear. The present study provides insights into the role that the negatively charged Asp325 residue may play in this context, at the bottom of FPN1 TM7b.…”

“…Pan et al thus proposed a transport scheme in TsFpn where H + and Fe 2+ share binding sites but do not occupy them simultaneously. In their model, each transport cycle exports one Fe 2+ that binds sequentially to the N-lobe (in a site containing the Asp39 and His43 residues) and the C-lobe (in a site containing the Cys326 and His508 residues) and imports two H + that occupy both sites [21]. However, it is not necessary for protonation to be fixed at two identical positions during the OF-to-IF transition, provided that there is one of the titratable residues inside the central cavity assuming the protonation status at any given time.…”

“…In order to get further insight into the role of Asp325 during the iron transport cycle, we first analyzed 3D structures in primate proteins solved in the OF state (Figure 3; panels A to C). Asp325 is not a direct ligand of the metal, either in TsFpn in presence of Co 2+ (pdb 6VYH, Figure 3A; [21]), or in HsFPN1 in presence of Co 2+ and hepcidin (pdb 6WBV, Figure 3B; [20]). Cys326 and His508(TsFpn)/His507(HsFPN1) are directly involved in the coordination of the metal, with atoms from the N-and C-terminus of hepcidin and water molecules providing additional ligands in the ferroportin-hepcidin complex to fulfill a tetrahedral coordination geometry (Figure 3B).…”

“…The authors confirmed the existence of two metal ion (Co 2+ )-binding sites. Using proteoliposomes, they further showed that Fe 2+ export is coupled to transport of H + [21].…”