Mika Jormakka

Iron is a vital trace element for almost all organisms, and maintaining iron homeostasis is critical for human health. In mammals, the only known gatekeeper between intestinally absorbed iron and circulatory blood plasma is the membrane transporter ferroportin (Fpn). As such, dysfunction of Fpn or its regulation is a key driver of iron-related pathophysiology. This review focuses on discussing recent insights from high-resolution structural studies of the Fpn protein family. While these studies have unveiled crucial details of Fpn regulation and structural architecture, the associated functional studies have also at times provided conflicting data provoking more questions than answers. Here, we summarize key findings and illuminate important remaining questions and contradictions.

Ferroportin (Fpn)—expressed at the plasma membrane of macrophages, enterocytes, and hepatocytes—mediates the transfer of cellular iron into the blood plasma. Under the control of the iron-regulatory hormone hepcidin, Fpn serves a critical role in systemic iron homeostasis. Although we have previously characterized human Fpn, a great deal of research in iron homeostasis and disorders uses mouse models. By way of example, the flatiron mouse, a model of classical ferroportin disease, bears the mutation H32R in Fpn and is characterized by systemic iron deficiency and macrophage iron retention. The flatiron mouse also appears to exhibit a manganese phenotype, raising the possibility that mouse Fpn serves a role in manganese metabolism. At odds with this observation, we have found that human Fpn does not transport manganese, so we considered the possibility that a species difference could explain this discrepancy. We tested the hypothesis that mouse but not human Fpn can transport manganese and performed a comparative analysis of mouse and human Fpn. We examined the functional properties of human Fpn, mouse Fpn, and mutant mouse Fpn by using radiotracer assays in RNA-injected Xenopus oocytes. We found that neither mouse nor human Fpn transports manganese. Mouse and human Fpn share identical properties with respect to substrate profile, calcium dependence, optimal pH, and hepcidin sensitivity. We have also demonstrated that Fpn is not an ATPase pump. Our findings validate the use of mouse models of ferroportin function in iron homeostasis and disease.

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.

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.

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.

In vertebrates, the iron exporter ferroportin releases Fe²⁺ from cells into plasma, thereby maintaining iron homeostasis. The transport activity of ferroportin is suppressed by the peptide hormone hepcidin, which exhibits upregulated expression in chronic inflammation, causing iron-restrictive anaemia. However, due to the lack of structural information about ferroportin, the mechanisms of its iron transport and hepcidin-mediated regulation remain largely elusive. Here we report the crystal structures of a putative bacterial homologue of ferroportin, BbFPN, in both the outward- and inward-facing states. Despite undetectable sequence similarity, BbFPN adopts the major facilitator superfamily fold. A comparison of the two structures reveals that BbFPN undergoes an intra-domain conformational rearrangement during the transport cycle. We identify a substrate metal-binding site, based on structural and mutational analyses. Furthermore, the BbFPN structures suggest that a predicted hepcidin-binding site of ferroportin is located within its central cavity. Thus, BbFPN may be a valuable structural model for iron homeostasis regulation by ferroportin.

GDP release from GTPases is usually extremely slow and is in general assisted by external factors, such as association with guanine exchange factors or membrane-embedded GPCRs (G protein-coupled receptors), which accelerate the release of GDP by several orders of magnitude. Intrinsic factors can also play a significant role; a single amino acid substitution in one of the guanine nucleotide recognition motifs, G5, results in a drastically altered GDP release rate, indicating that the sequence composition of this motif plays an important role in spontaneous GDP release. In the present study, we used the GTPase domain from EcNFeoB (Escherichia coli FeoB) as a model and applied biochemical and structural approaches to evaluate the role of all the individual residues in the G5 loop. Our study confirms that several of the residues in the G5 motif have an important role in the intrinsic affinity and release of GDP. In particular, a T151A mutant (third residue of the G5 loop) leads to a reduced nucleotide affinity and provokes a drastically accelerated dissociation of GDP.

GTPases (G proteins) hydrolyze the conversion of GTP to GDP and free phosphate, comprising an integral part of prokaryotic and eukaryotic signaling, protein biosynthesis and cell division, as well as membrane transport processes. The G protein cycle is brought to a halt after GTP hydrolysis, and requires the release of GDP before a new cycle can be initiated. For eukaryotic heterotrimeric G_αβγ proteins, the interaction with a membrane‐bound G protein‐coupled receptor catalyzes the release of GDP from the G_α subunit. Structural and functional studies have implicated one of the nucleotide binding sequence motifs, the G5 motif, as playing an integral part in this release mechanism. Indeed, a G_αs G5 mutant (A366S) was shown to have an accelerated GDP release rate, mimicking a G protein‐coupled receptor catalyzed release state. In the present study, we investigate the role of the equivalent residue in the G5 motif (residue A143) in the prokaryotic membrane protein FeoB from Streptococcus thermophilus, which includes an N‐terminal soluble G protein domain. The structure of this domain has previously been determined in the apo and GDP‐bound states and in the presence of a transition state analogue, revealing conformational changes in the G5 motif. The A143 residue was mutated to a serine and analyzed with respect to changes in GTPase activity, nucleotide release rate, GDP affinity and structural alterations. We conclude that the identity of the residue at this position in the G5 loop plays a key role in the nucleotide release rate by allowing the correct positioning and hydrogen bonding of the nucleotide base.

FeoB is a transmembrane protein involved in ferrous iron uptake in prokaryotic organisms. FeoB comprises a cytoplasmic soluble domain termed NFeoB and a C-terminal polytopic transmembrane domain. Recent structures of NFeoB have revealed two structural subdomains: a canonical GTPase domain and a five-helix helical domain. The GTPase domain hydrolyses GTP to GDP through a well characterized mechanism, a process which is required for Fe²⁺transport. In contrast, the precise role of the helical domain has not yet been fully determined. Here, the structure of the cytoplasmic domain of FeoB fromGallionella capsiferriformansis reported. Unlike recent structures of NFeoB, theG. capsiferriformansNFeoB structure is highly unusual in that it does not contain a helical domain. The crystal structures of both apo and GDP-bound protein forms a domain-swapped dimer.