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Structural basis of ferroportin inhibition by minihepcidin PR73 [1]

['Azaan Saalim Wilbon', 'Verna', 'Marrs Mclean Department Of Biochemistry', 'Molecular Biology', 'Baylor College Of Medicine', 'Houston', 'Texas', 'United States Of America', 'Jiemin Shen', 'Piotr Ruchala']

Date: 2023-01 

Abstract Ferroportin (Fpn) is the only known iron exporter in humans and is essential for maintaining iron homeostasis. Fpn activity is suppressed by hepcidin, an endogenous peptide hormone, which inhibits iron export and promotes endocytosis of Fpn. Hepcidin deficiency leads to hemochromatosis and iron-loading anemia. Previous studies have shown that small peptides that mimic the first few residues of hepcidin, i.e., minihepcidins, are more potent than hepcidin. However, the mechanism of enhanced inhibition by minihepcidins remains unclear. Here, we report the structure of human ferroportin in complex with a minihepcidin, PR73 that mimics the first 9 residues of hepcidin, at 2.7 Å overall resolution. The structure reveals novel interactions that were not present between Fpn and hepcidin. We validate PR73-Fpn interactions through binding and transport assays. These results provide insights into how minihepcidins increase inhibition potency and will guide future development of Fpn inhibitors.

Citation: Wilbon AS, Shen J, Ruchala P, Zhou M, Pan Y (2023) Structural basis of ferroportin inhibition by minihepcidin PR73. PLoS Biol 21(1): e3001936. https://doi.org/10.1371/journal.pbio.3001936 Academic Editor: Raimund Dutzler, University of Zurich, SWITZERLAND Received: September 1, 2022; Accepted: November 30, 2022; Published: January 17, 2023 Copyright: © 2023 Wilbon et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: The cryo-EM density maps of nanodisc-encircled HsFpn-11F9 bound to PR73 or Co2+ have been deposited in the Electron Microscopy Data Bank (https://www.ebi.ac.uk/pdbe/emdb/) under accession codes EMD-27498 and EMD-27499, respectively. The corresponding atomic coordinate files have been deposited in the Protein Data Bank (http://www.rcsb.org) under ID codes 8DL7 (HsFpn-PR73) and 8DL8 (HsFpn-Co2+). Source data are available in S1 Data. Funding: This work was supported by grants from the National Heart, Lung, and Blood Institute (HL157473 to Y.P.), National Institute of Diabetes and Digestive and Kidney Diseases (DK122784 to M.Z.), National Institute of General Medical Sciences (GM145416 to M.Z), National Heart, Lung, and Blood Institute (HL086392 to M.Z.) and Cancer Prevention and Research Institute of Texas (R1223 to M.Z.). Cryo-EM data in this work were acquired at the Pacific Northwest Center for Cryo-EM (PNCC) at Oregon Health & Science University, supported by the National Institute of General Medical Sciences grant U24GM129547, and the National Center for Cryo-EM Access and Training (NCCAT) and the Simons Electron Microscopy Center at the New York Structural Biology Center, supported by the National Institute of General Medical Sciences grant U24GM129539 and by grants from the Simons Foundation (SF349247) and NY State Assembly. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: AH, amphipathic helix; BLI, biolayer interferometry; BME, β-mercaptoethanol; CTD, C-terminal domain; cryo-EM, cryo-electron microscopy; Fab, fragment of antigen binding; Fpn, ferroportin; HEK, human embryonic kidney; LCIS, live cell imaging solution; MSP, membrane scaffold protein; NTD, N-terminal domain; NU, non-uniform; RMSD, root-mean-squared distance; SEC, size exclusion chromatography; TEV, tobacco etch virus; TM, transmembrane; WT, wild type

Introduction Ferroportin (Fpn) is a Fe2+/2H+ antiporter that is highly expressed in enterocytes, hepatocytes, and macrophages to export Fe2+ derived from either dietary intake or digestion of senescent red blood cells [1–3]. Since Fpn is the only known iron exporter in mammals, its activity is essential for plasma iron homeostasis [4]. Ferroportin activity can be acutely suppressed by the peptide hormone hepcidin that binds to Fpn to inhibit iron transport. Binding of hepcidin also induces endocytosis and degradation of Fpn to further reduce iron transport [5,6]. Meanwhile, the expression of hepcidin is regulated via plasma iron levels, resulting a closely monitored hepcidin-ferroportin axis [7,8]. Mutations in Fpn that impair transport activity can cause ferroportin disease, leading to symptoms of iron-deficiency anemia [9,10]. Elevated hepcidin levels can also lead to iron deficiency [11]. Hepcidin deficiency and hepcidin-resistant mutations in Fpn, on the other hand, lead to hereditary hemochromatosis and iron overload [10]. Thus, the hepcidin-ferroportin axis must be tightly regulated to maintain serum iron levels. Hepcidin is a peptide of 25 amino acids and is secreted by hepatocytes. Hepcidin binds to the extracellular side of Fpn [2,12,13]. There are 4 pairs of intramolecular disulfide bridges in hepcidin [14,15] (Fig 1A), and mutational studies showed that the first 7–9 residues have a large impact on its ability to inhibit Fpn [16]. The dissociation of the Fpn-hepcidin complex under reducing conditions led to the hypothesis that the disulfide bridge between Cys7 and Cys23 in hepcidin could be replaced with a disulfide bridge between Cys7 and Cys326 on Fpn [17], although this exchange was not observed in the structure of Fpn bound to hepcidin [12]. These studies led to the development of a new class of potent Fpn inhibitors, termed minihepcidins, that are based on the first 7–9 amino acids of hepcidin. Some of the minihepcidins have drastically improved potency to Fpn and have been further optimized and tested as hepcidin replacements to treat patients with dysregulation of hepcidin [18]. However, it remains unknown how the minihepcidins bind to Fpn with a higher affinity than hepcidin. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Structural comparison of hepcidin and PR73 molecules. (a) Structure of hepcidin in complex with HsFpn (PDB: 6WBV). Hepcidin (marine), HsFpn (light blue), and Co2+ (light pink) are shown in the overall (inset) and zoomed-in views. Side chains of the first seven residues and all cysteines in hepcidin are shown as sticks, and so is the carboxyl group of the last residue. The hepcidin sequence is shown as single-letter codes at the bottom, and the disulfide bridges are marked by black lines. (b) Chemical structure of PR73. Unnatural amino acid abbreviations are as follows: Ida, iminodiacetic acid; Dpa, diphenylalanine; bhPro, beta homo-proline; bhPhe, Beta homo-phenylalanine; Ahx, aminohexanoic linker. https://doi.org/10.1371/journal.pbio.3001936.g001 Structures of Fpn in complex with hepcidin revealed how the 2 interact [2,12]. Hepcidin binds between the N-terminal domain (NTD) and C-terminal domain (CTD) of Fpn, and the first 6 residues of hepcidin interact with Fpn. Hepcidin retains all 4 of its internal disulfide bonds, and its C-terminal carboxylate coordinates 1 metal ion at one of the binding sites, which is formed by Cys326 and His507 of Fpn [12]. Importantly, residue Cys326 is known to affect hepcidin binding and is associated with hemochromatosis [17,19]. These structures have presented insights into the binding mechanism of hepcidin to Fpn. Minihepcidins are highly effective in inhibiting Fpn activity [20]. PR73 is a minihepcidin and a highly potent inhibitor of Fpn [21] whose predecessor showed effectiveness in preventing iron overload [18]. PR73 mimics the first 9 residues of hepcidin, including the cysteine in position 7 that was hypothesized to form a disulfide bridge with residue Cys326 of Fpn [17] (Fig 1B). The last 16 residues of hepcidin have been replaced with an aminohexanoic linker and iminodiacetic palmitic amide (Ida(NHPal)) in PR73, eliminating the C-terminal carboxylate essential for the binding of hepcidin. Thus, it is not clear how PR73 inhibits Fpn with increased potency. Here, we present the structures of human (Homo sapiens) Fpn (HsFpn) bound to PR73 and Co2+ at 2.7 Å and 3.0 Å, respectively. We used a fragment of antigen-binding (Fab) to facilitate structure determination by cryo-electron microscopy (cryo-EM). The structures show that PR73 preserves most of the original interactions between hepcidin and Fpn. Although PR73 lacks the C-terminal carboxylate to coordinate the bound metal ion, it forms a disulfide bridge with HsFpn, and the palmitic amide of PR73 interacts with Gln194 on Fpn, which positions the acyl chain of PR73 in the hydrophobic core of the cell membrane surrounding Fpn.

Discussion Here, we report a study on 2 inhibitors of HsFpn. First, we showed that mouse monoclonal 11F9 Fab, which was developed to bind to a monkey homolog of Fpn, binds to HsFpn with nanomolar affinity and inhibits ion transport. We determined the structure of HsFpn in complex with 11F9 Fab and found that the Fab interacts with both the NTD and CTD of HsFpn from the intracellular side, and the interactions likely stabilize the transporter in an outward-facing conformation. Second, we identified critical components of PR73-HsFpn interactions. We determined the structure of HsFpn in complex with PR73, and we find that the first 6 residues of PR73 assume a similar conformation to that of hepcidin, even though 3 of the 6 residues in PR73 are modified to unnatural amino acids, and that these residues interact with HsFpn similar to hepcidin. However, residues 7–11 of PR73 interact with HsFpn differently. While hepcidin has a compact and well-folded structure with 4 disulfide bridges, PR73 has none. The free Cys7 of PR73 could form a disulfide bond to Cys326 of HsFpn (Fig 8A), and we show that the interaction contributes significantly to the binding of PR73. Although it was proposed previously that hepcidin form a disulfide bridge with HsFpn, the structure of hepcidin-HsFpn shows that its internal cysteine bridges are intact (Fig 8B). The structure also shows that the flexibility of PR73 affords the interaction between residue 11 of PR73 and Gln194 of HsFpn, and we show that this interaction also contributes significantly to the binding. Finally, the structure shows that the hydrophobic acyl chain of the palmitic acid may extend into the hydrophobic core of the membrane to anchor PR73 in the membrane and thus may enhance its binding affinity. PPT PowerPoint slide

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TIFF original image Download: Fig 8. Structural comparison of PR73 and hepcidin-bound HsFpn. Side views of HsFpn-PR73 (a) and HsFpn-Hepcidin (6WIK) (b) highlight the disulfide bridge and metal ion coordination. In (a), TM7b is colored pale green and PR73 in brick red. In (b), TM7b is colored in light blue, hepcidin shown as marine cartoon with cysteine residues, and C-terminal carboxylate shown as sticks. https://doi.org/10.1371/journal.pbio.3001936.g008 Based on the structure of PR73 in complex with HsFpn, and considering that extracellular redox potential is usually in the range permissible for disulfide bridge formation, we think that Cys7 of PR73 would form a disulfide bridge with Cys326 of HsFpn, which could significantly enhance the inhibition. Likewise, the stability of hepcidin is also dependent on formation of stable disulfide bridges and we surmise that under normal physiological conditions, hepcidin is well folded and optimized to inhibit HsFpn. If macrophages or hepatocytes encounter conditions that do not favor stable disulfide bridge formation, then inhibition of Fpn by either hepcidin or PR73 would be compromised. Minihepcidins are promising therapeutic reagents being pursued for the treatment of human diseases like β-thalassemia and hemochromatosis [16,18]. Our study provides a structural framework that highlights unique interactions between PR73 and Fpn and may facilitate and guide future drug development targeting HsFpn.

Materials and methods Cloning, expression, and purification of HsFpn The cDNA of HsFpn (UniProt ID: Q9NP59) was codon optimized, synthesized, and cloned into a pFastBac dual vector. A tobacco etch virus (TEV) protease site and an octa-histidine (8×His) tag were added to the C-terminus of the protein. The Back-to-Bac method (Invitrogen) was used to express HsFpn was expressed in Sf9 (Spodoptera frugiperda). Purification of HsFpn follows the same protocol reported for TsFpn [2]. Size exclusion chromatography (SEC) was used to collect the purified HsFpn in FPLC buffer consisting of 20 mM HEPES (pH7.5), 150 mM NaCl, and 1 mM (w/v) n-dodecyl-β-D-maltoside (DDM, Anatrace). The Quikchange method (stratagene) was used to generate HsFpn mutations. Mutations were verified by sequencing. Mutant HsFpn proteins were expressed and purified following the same protocol for the WT. Octet biolayer interferometry Biolayer interferometry (BLI) assays were performed at 30°C under constant shaking at 1,000 rpm using an Octet system (FortéBio). First, amine-reactive second-generation (AR2G) biosensor (Sartorius) tips were activated in 20 mM 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) and 10 mM N-hydroxysulfosuccinimide (Sulfo-NHS) for 300 s. Then, the tips were immobilized with 5 μg/mL of 11F9 Fab in the FPLC buffer for 600 s. The tips were quenched in 1 M ethanolamine (pH 8.5) for 300 s. The tips with immobilized ligands were equilibrated in the FPLC buffer for 120 s and transferred to wells with a concentration gradient of HsFpn (400, 200, 100, 50, and 25 nM) for 300 s (association) and returned to the equilibration wells for dissociation (300 s). To measure PR73 binding, the tips were immobilized with Fpn at a concentration of 2 μg/mL in the FPLC buffer for 600 s. After quenching the immobilization reaction, the tips were transferred to wells with a concentration gradient of PR73 (10, 5, 2.5, 1.25 μM) for 300 s (association) and back to equilibration wells for 300 s (dissociation). Binding curves were aligned and corrected with the channel of no analyst protein. The association and disassociation phases were fit with 1-exponential functions to extract association rate constant k a and dissociation rate constant k d of the binding, which were used to calculate the dissociation constant K D . Reconstitution of Fpn into liposomes The 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac)-ethanolamine (POPE, Avanti Polar Lipids) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1’-rac)-glycerol (POPG, Avanti Polar Lipids) was mixed at 3:1 molar ratio, dried with Argon, and vacuumed overnight to remove chloroform. The lipid was resuspended in reconstitution buffer (20 mM HEPES (pH 7.5), 100 mM NaCl) to a final concentration of 10 mg/mL. The lipid was sonicated until it appeared transparent. A total of 40 mM n-decyl-β-D-maltoside (DM, Anatrace) was added and the sample was incubated for 2 h at room temperature under gentle agitation. HsFpn was added at a 1:100 (w/w, protein:lipid) ratio. Dialysis was performed at 4°C with the reconstitution buffer to remove the detergent. The dialysis buffer was changed daily for 3 days and then harvested on day 4. Liposome samples were aliquoted and frozen at −80°C for future use. Co2+ and Fe2+ flux assays in proteoliposomes Proteoliposome samples of HsFpn were mixed with 250 μM calcein, with or without 20 μM 11F9 Fab, and underwent 3 cycles of freeze-thaw. The liposomes were extruded to homogeneity with a 400 nm filter (NanoSizer Extruder, T&T Scientific Corporation). Excess calcein was removed with a desalting column (PD-10, GE Healthcare) that had been equilibrated with the dialysis buffer. A quartz cuvette was used to detect the fluorescence at 37°C. For the samples loaded with the Fab, an additional 20 μM of 11F9 Fab was incubated with the liposomes prior to reading. The cuvette was read at 10 s intervals with 494 nm excitation and 513 nm emission in a FluoroMax-4 spectrofluorometer (HORIBA). Approximately 100 μM CoCl 2 or 100 μM Fe2+ (together with 1 mM ascorbate-Na) was added to initiate transport. Preparation of Fpn-11F9 complex in nanodisc An established protocol [25] was used to express and purify membrane scaffold protein (MSP) 1D1. Lipid preparation was carried out by mixing 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac)-choline (POPC, Avanti Polar Lipids), POPE, and POPG at a molar ratio of 3:1:1. The lipid mixture was dried with Argon and vacuumed for 2 h. The lipid was resuspended with 14 mM DDM [26]. HsFpn, MSP1D1, and the lipid mixture were mixed at a molar ratio of 1:2.5:50 and incubated on ice for 1 h for nanodisc reconstitution. A total of 60 mg/mL of Biobeads SM2 (Bio-Rad) were added 3 times within 3 h to remove detergents. After the samples were incubated with the Biobeads overnight at 4°C, the Biobeads were removed. 11F9 Fab was added to the nanodisc sample at a molar ratio of 1.1:1 to Fpn. The complex was incubated on ice for 30 min before it was loaded onto a SEC column that had been equilibrated with 20 mM HEPES (pH 7.5) and 150 mM NaCl. The purified nanodisc sample was concentrated to 10 mg/ml and incubated with 10 mM CoCl 2 or 1 mM PR73 for 30 min before cryo-EM grid preparation. Cryo-EM sample preparation and data collection The cryo-EM grids were prepared with Thermo Fisher Vitrobot Mark IV. The Quantifoil R1.2/1.3 Cu grids were glow-discharged with air at 10 mA for 15 s using Plasma Cleaner (PELCO EasiGlow). Aliquots of 3.5 μL of the nanodisc sample were applied to the glow-discharged grids. The grids were blotted with filter paper (Ted Pella) for 4.0 s and plunged into liquid ethane cooled with liquid nitrogen. A total of 4,251 (for Co2+-bound Fpn) and 4,941 (for PR73-bound Fpn) micrograph stacks were collected on a Titan Krios at 300 kV equipped with a K3 Summit direct electron detector (Gatan) and a Quantum energy filter (Gatan) at a nominal magnification of 81,000× and defocus values from −2.25 to −1.0 μM (HsFpn-Co2+) or −2.5 to −0.8 μM (HsFpn-PR73). Each stack was exposed for 0.0875 s per frame in the super-resolution mode for a total of 40 frames per stack, which results in a total dose of approximately 50 e-/Å2. The stacks were motion corrected with MotionCor2 [27] and binned 2-fold. The final pixel size is 1.08 Å/pixel (HsFpn-Co2+) or 1.10 Å/pixel (HsFpn-PR73). In the meantime, dose weighting was performed [28]. The defocus values were estimated with Gctf [29]. Cryo-EM data processing A total of 2,175,353 (HsFpn-Co2+) and 2,960,056 (HsFpn-PR73) particles were automatically picked based on a reference map of TsFpn-11F9 (EMD-21460) that was low-pass filtered to 20 Å in RELION 3.1 [30–32]. Particles were extracted and imported into CryoSparc [33] for 2D classification. A total of 1,242,825 (HsFpn-Co2+) and 918,469 particles (HsFpn-PR73) were selected from good classes in 2D classification. A total of 100,000 particles for the good classes were used to generate 4 initial reference models. Multiple rounds of heterogeneous refinement were performed with particles selected from the 2D classification until <5% input particles were classified into bad classes. A total of 215,164 particles (HsFpn-Co2+) or 454,601 particles (HsFpn-PR73) were subjected to non-uniform (NU) refinement. After handedness correction, local refinement and CTF refinement were performed, resulting in a reconstruction with an overall resolution of 3.0 Å for HsFpn-Co2+ and 2.6 Å for HsFpn-PR73. Additional rounds of heterogenous refinement were performed for HsFpn-PR73 with 3 reference models of “class similarity” of 1 to further improve the density for PR73. Classes with strong densities for PR73 were selected and subjected to NU refinement. The final total of 162,586 particles yielded a reconstruction with an overall resolution of 2.7 Å for HsFpn-PR73. Resolution was estimated with the gold-standard Fourier shell correlation 0.143 criterion [34]. Local resolution of the maps was estimated in CryoSparc [33]. Model building and refinement The structure of apo HsFpn (PDB ID 6W4S) and the 11F9 Fab (from PDB ID 6VYH) were individually docked into density maps in Chimera [35]. The docked model was manually adjusted in COOT [36]. PHENIX [37] was used for real space refinements with secondary structure and geometry restraints. The EMRinger Score [38] was calculated for the models. Structure figures were prepared in Pymol and ChimeraX [39]. Fe2+, H+, and Ca2+ transport assays in HEK cells The pEG BacMam plasmids with WT or mutant Fpn or the empty plasmid were transfected into HEK 293S cells on black wall 96-well microplates coated with poly-D-lysine (Invitrogen/Thermo Fisher). After 2 days, cells were washed in the live cell imaging solution (LCIS) containing 20 mM HEPES (pH 7.4), 140 mM NaCl, 2.5 mM KCl, 1.0 mM MgCl 2 , and 5 mM D-glucose. The loading of PhenGreen FL (Invitrogen/Thermo Fisher, Diacetate) for Fe2+ transport, pHrodo Red (Invitrogen/Thermo Fisher, AM) for H+ transport, or Fluo-4 (Invitrogen/Thermo Fisher, AM, cell-permeant) for Ca2+ transport was performed following the manufacturer’s protocols. After the dye loading finished, free dyes were washed away, and cells in each well were maintained in 90 μL LCIS. All transport assays were performed in the FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) at 37°C. Fluorescence changes were recorded at an excitation and emission wavelength of 492 nm and 517 nm for Fe2+ transport, 544 nm and 590 nm for H+ transport, or 485 nm and 538 nm for Ca2+ transport with 5 s intervals. For PR73 inhibition, cells were incubated with desired concentrations of PR73 for 5 min prior to reading. Transport was initiated by the addition of 10 μL freshly prepared ligand stock solution to achieve 100 μM Fe2+ (together with 1 mM ascorbate-Na) or 500 μM Co2+ or Ca2+. For Fe2+ and H+ transport, relative fluorescence changes at the equilibrium stage were averaged to represent intracellular [Fe2+] or pH changes. For Ca2+ uptake, the slopes of straight lines fitted to transport data within 25 s were used to represent initial rates.

Acknowledgments We acknowledge the use of the cryo-EM core at Baylor College of Medicine (BCM) for grid preparation and screening. We acknowledge L. Wang for help with grid preparation, and Z. Ren for making some of the mutations. We are grateful to A. A. R. Adeosun for her instructions on the use of FlexStation 3.

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