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An improved structural model of the human iron exporter ferroportin. Insight into the role of pathogenic mutations in hereditary hemochromatosis type 4

An improved structural model of the human iron exporter ferroportin. Insight into the role of... IntroductionIron is the second most abundant metal on Earth’s crust and plays an essential biological role in all living organisms [1]. Biological properties of iron are linked to its efficient electron transfer properties, allowing it to accept or donate electrons. This ability enables it not only to participate as a catalyzing cofactor in various biochemical reactions but also to contribute to the formation of potentially damaging free radical species [2]. Thus, iron concentration in body tissues needs to be strongly regulated. In fact, it is well known that the imbalance of iron homeostasis has noticeable effects on human health [1].Ferroportin (Fpn) is a membrane protein representing the major cellular iron exporter, essential for metal translocation from cells into plasma [3]. It belongs to one of the largest secondary transporter families, the major facilitator superfamily (MFS), and together with hepcidin, a 25-amino-acid peptide that binds to hFpn and drives its degradation, it is a key player in the control of human iron homeostasis [3]. Mutations in the gene coding for hFpn cause type 4 hemochromatosis [4], an autosomal dominant iron overload condition. Mutations are generally classified into two subclasses: “loss-of-function” and “gain-of-function”. The majority of pathogenic variants are defined as “loss-of-function,” as they lead to decreased iron export from cells. These give rise to type 4A hemochromatosis, also known as Fpn disease [5]. On the other hand, “gain-of-function” mutations, responsible for type 4B hemochromatosis, confer resistance to the action of hepcidin. Hepcidin binding to Fpn leads to protein internalization, ubiquitination, and degradation. Gain-of-function mutations are named this way because they inhibit this mechanism and cause Fpn to permanently stay on the membrane and continue to be active in iron efflux.Although the importance of Fpn in human iron homeostasis has been widely demonstrated, its exact three-dimensional structure remains unknown. Based on the knowledge of the structure of other members of the MFS family, it was proposed that Fpn operates through an alternating-access mechanism that involves a movement of the two halves of the protein, together with local structural rearrangements. The rotation of the two domains composing the protein leads to three different conformational states: outward-facing, occluded, and inward-facing [6, 7]. Despite the lack of a crystal structure, different models of hFpn have been proposed. The first available hFpn model [8] can be considered a “low-resolution” one. In fact, the transmembrane helices were predicted and built de novo and then manually fitted over the three-dimensional structure of the Escherichia coli glycerol-3-phosphate transporter in the inward-facing conformation (PDB code: 1PW4 [9]). A few years later, Le Gac and coworkers [10] described a novel model obtained using the three-dimensional structure of the MFS transporter EmrD (PDB code: 2GFP [11]) in the occluded conformation. However, loops outside of the transmembrane helices were not modeled, and some critical structural details of this model were later shown to be incorrect. For instance, Asp181, a residue affected in loss-of-function mutants, was hypothesized to be exposed toward the membrane while it was later shown to be essential for iron binding and transport and thus exposed within the substrate-binding pocket [12]. The first structural models of hFpn in both the inward- and outward-facing conformations were later obtained by Bonaccorsi di Patti and coworkers [12] on the basis of the three-dimensional structures of the glycerol-3-phosphate transporter (PDB code:1PW4 [9]) and the FucP fucose transporter (PDB code: 3O7Q [13]), respectively. These models allowed to uncover and experimentally validate the identity of residues involved in iron binding and transport. Finally, a possible iron-transport mechanism was hypothesized on the basis of hFpn models in all three mechanistically relevant conformations (inward-facing, occluded, and outward-facing) [7].Recently, a bacterial homologue of hFpn has been identified in the predatory bacterium Bdellovibrio bacteriovorus [14, 15] and the three-dimensional structure of this protein (BdFpn) has been determined in the inward- and outward-facing conformational states. Taking advantage of the availability of these structures, the only ones available for a member of the Fpn family, updated models of hFpn in the inward- and outward-facing conformations have been built and a comprehensive structure-function analysis of known Fpn pathogenic mutations has been carried out.MethodsMolecular modelingThe structural models of hFpn have been built using the ab initio/threading approach implemented in the I-TASSER pipeline [5] and the homology modeling software packages MODELLER [16] and SwissModel [17]. In the case of I-TASSER and SwissModel, no query/template alignment has been provided in input. In fact, in the case of I-TASSER, the server uses LOMETS (Local Meta-Threading Server) to thread the query sequence through a representative PDB structure library to search for the possible folds compatible with the sequence of the protein of interest [18, 19]. SwissModel instead uses PSI-BLAST [20] to construct the best query-template alignment. In the case of MODELLER, a reliable sequence alignment between hFpn and BdFpn has been given in input. This was obtained using CLUSTAL-omega [21] to align the amino acid sequence (NCBI GI code reported within parentheses) of BdFpn (gi 426403971) to those of Fpn from Homo sapiens (gi 7657100), Bos taurus (gi 118151032), Rattus norvegicus (gi 158635998), Mus musculus (gi 7109247), Anas platyrhynchos (gi 483522646), Gallus (gi 61098366), Chelonia mydas (gi 465955074), Danio rerio (gi 7109245), Echinops telfairi (gi 507641023), Tetraodon nigroviridis (gi 47225539), Branchiostoma floridae (gi 229294181), Caenorhabditis elegans (gi 351061132) and Ciona intestinalis (gi 198437857). In all three cases, modeling has been carried out by specifying as templates the three-dimensional structures of BdFpn in the inward-facing (PDB code: 5AYO [14]) and outward-facing (PDB code: 5AYM [14]) conformation. The overall quality of the models has been assessed using PROCHECK [22], and the corresponding G-factor values (the PROCHECK G-factor threshold for good-quality three-dimensional structures being −0.5) are reported in Supplementary Materials Table S1.Structure-based sequence alignment of the models to the three-dimensional structure of BdFpn in the inward-facing and outward-facing conformations has been obtained using ESPript 3.0 [23].Docking simulationsDocking simulations between the three-dimensional structure of hepcidin (PDB code: 3H0T [24]) and the structural model of hFpn in the outward-facing conformation have been performed using the protein-protein docking server ZDOCK (version 3.0.2), which is based on a Fast Fourier transform algorithm and a scoring system combining shape complementarity, electrostatics and statistical potential terms [25]. The 2000 complexes predicted by ZDOCK were re-ranked using ZRANK, which uses a more detailed potential combining electrostatics, van der Waals and desolvation terms [26].Collection of pathogenic mutationsA list of all known hFpn pathogenic mutations has been obtained through a careful manual analysis of the available literature. Most mutations were retrieved from the comprehensive meta-analysis by Mayr et al. [27]; the list has recently been updated [5, 27].Results and discussionAnalysis of the structural models of hFpn in the inward- and outward-facing conformationsThe structural models of hFpn in the inward- and outward-facing conformations obtained with I-TASSER, MODELLER, and SwissModel are shown in Figure 1. To further assess the degree of agreement between the different models at the residue level, a structure-based sequence alignment has been carried out. As can be seen in Supplementary Materials Figures S1 and S2, the three different models display a high degree of consensus in the core structure (transmembrane helices) while, for obvious reasons, large deviations are observed at the level of the intracellular and extracellular surface loops. From this viewpoint, it must be noted that in the models obtained by homology modeling, the surface loops are not structured. On the contrary, the threading/ab initio approach implemented in I-TASSER, including replica exchange molecular dynamics simulations, allows to predict a putative structure of the large loops, which characterize hFpn. In other structurally characterized members of the MFS, these loops are involved in the inward to outward conformational change [28]. Therefore, once assessed, the substantial agreement between the three methods, the subsequent structural analyses were conducted on the hFpn models generated with I-TASSER.Figure 1:Schematic representation of the structural models of hFpn obtained in this study.Structural models of hFpn obtained with I-TASSER (tan), MODELLER (cyan), and SwissModel (purple) in the inward-facing (A) and outward-facing (B) conformations.The superposition of BdFpn structures with the hFpn models generated by I-TASSER yields RMSD values of 1.24 Å for the inward-facing state and of 1.06 Å for the outward-facing state, with the most noticeable differences being located at the level of the TM2-TM3 connecting loop. This difference is acceptable, as this loop is considerably longer in hFpn with respect to BdFpn, and has been subjected to an ab initio modeling procedure in the I-TASSER workflow.As already emerged from previous studies [7, 8, 10, 12, 14], the structural model of hFpn consists of 12 transmembrane helices (TM), which are arranged in two domains (N-domain and C-domain), each containing six consecutive transmembrane helices, surrounding a substrate translocation pore, with the N- and C-termini located on the intracellular side. Helices, 1, 2, 4, 5, 7, 8, 10 and 11 shape the central transport cavity, and helices 3, 6, 9 and 12 form the outer domain.Experimental studies carried out on hFpn have identified functional roles for selected residues in iron binding and transport. In agreement with the biochemical characterization of other MFS transporters, the hypothetical substrate-binding site of hFpn is located halfway into the membrane [6], with the TM7 contributing more than half of the interacting residues for ligand coordination. In detail, Asp39, Asp181 and Asp325, whose mutation completely abolishes the iron binding and transport ability [12], form a putative iron binding site (Figure 2); Arg466 mutation significantly reduces the iron transport ability of Fpn and has been hypothesized to act as an “electrostatic switch”, which, upon iron binding, facilitates the inward to outward transition of Fpn [7]. These residues are conserved in BdFpn, with the exception of hFpn Asp325, which substitutes His261 present in the orthologous position in BdFpn [15]. Unexpectedly, mutation of BdFpn His261 into Asp (mimicking hFpn in this regard) completely abolished iron transport [15]. Analysis of the latest inward-facing model of hFpn provides a likely explanation for this result. In fact, Asp325 is predicted to be located in the vicinity of His43 (Figure 2), and a compensative substitution of hFpn His43 with Asp28 in BdFpn is observed, indicating a functional role of an Asp-His pair (Asp325-His43 in hFpn; Asp28-His261 in BdFpn), which is disrupted in the BdFpn His261Asp mutant. Thus, the two Fpns contain similarly oriented His-Asp pairs, and this observation, together with the reduced iron transport ability observed upon mutation of Asp325 in hFpn [12] and His261 in BdFpn [15], indicates that this residues pair likely plays a role in iron binding and/or translocation. The overall structure of Fpn shows that the transporter consists of irregular, highly kinked transmembrane helices that could be stabilized by the hydrophobicity and/or size of the amino acid side chains. Several “loss-of-function” mutations are located in positions that could induce irregularities in helices [29] indicating that, although none of these residues is required for hFpn activity, the bulkyness of the side chain at these positions could be important for conformational flexibility.Figure 2:Selected, functionally-relevant structural details of the hFpn structural models.Schematic representation of (A) the hFpn putative iron binding site viewed from the cytoplasmic side in the inward-facing conformation; (B) Motif-A involving Gly80, Asp84, Arg88 and Asp157 in the outward-facing conformation (Gly80 position indicated by the grey ribbon color); (C) the cytoplasmic kinked, amphipatic α-helix interacting with the cytoplasmic ends of the transmembrane helices in the outward-facing conformation.Another important motif identified in this molecule is Motif-A [28] (Figure 2), located at the level of the cytoplasmic loop connecting TM2 and TM3, formed by residues Gly80 (Gly65 in BdFpn), Asp84 (Asp69 in BdFpn) and Arg88 (Arg73 in BdFpn) [7, 15]. This motif, largely conserved in members of the MFS family, does not likely play a direct role in the iron transport mechanism but may only function as a cytoplasmic anchor point probably coordinating the conformational changes of MFS transporters by stabilizing the outward-facing conformation [6, 30].An interesting feature of the outward-facing model of hFpn is the presence, on the cytoplasmic side, of a kinked amphipathic α-helix seating at the cytoplasm-membrane boundary and interacting with the cytoplasmic ends of the transmembrane α-helices (Figure 2). This helix corresponds to the long loop connecting the N- and C-terminal domains in the inward-facing conformation. Interestingly, in other members of the MFS, the structuring of this loop into an amphipathic α-helix has been suggested to drive the inward- to outward-facing conformation [30].Mapping of all known mutations on the structural models of hFpnTable 1 shows a list of all hFpn mutations known up to date, together with the corresponding clinical and biochemical phenotypes. From this viewpoint, it must be mentioned that a structure-function analysis of a restricted subset of these mutations was already reported by Le Gac and coworkers [10] on the basis of the hFpn structural model in the occluded conformation. However, as briefly mentioned in the Introduction, some of the structural details of this model were later shown not to be consistent with experimental data and, given the occluded conformation of the model, the role of mutations conferring hepcidin resistance was difficult to assess.Table 1:Classification of hFpn missense variants.MutationClinical phenotypeBiochemical phenotypeY64N/HNon classicalGain of functionA69TNon classicalGain of functionS71FNon classicalGain of functionV72FNon classicalGain of functionA77DVariableLoss of functionG80S/VVariableLoss of functionD84EClassicalLoss of functionR88G/TClassicalLoss of functionL129PClassicalLoss of functionN144H/D/TVariableGain of functionI152FClassicalLoss of functionD157G/Y/NClassicalLoss of functionW158L/CClassicalLoss of functionV162delClassicalLoss of functionL170FN/ALoss of functionN174IVariableLoss of functionR178QVariableN/AI180T–NeutralD181V/NClassicalLoss of functionQ182HClassicalLoss of functionN185DVariableLoss of functionG204S/RVariableGain of functionT230N–NeutralL233PVariableLoss of functionK240ENon classicalGain of functionQ248H–SNPM266T–NeutralG267DN/AN/AD270VNon classicalGain of functionR296QClassicalLoss of functionG323VN/ALoss of functionF324S–SNPC326S/Y/FNon classicalGain of functionS338RNon classicalGain of functionG339D–SNPL345F–NeutralI351V–NeutralR371Q–NeutralL384M/V–SNPF405S–SNPM432V–SNPP443L–SNPR489S/KVariableLoss of functionG490D/SVariableLoss of functionY501CNon classicalGain of functionD504NNon classicalGain of functionH507RNon classicalGain of functionR561G–SNPSNP, single nucleotide polymorphism; N/A, not available.Here we report a detailed analysis of the putative structural-functional role of the gain-of-function and loss-of-function hFpn mutations based on the novel models obtained. A large part of the mutations is located on transmembrane helices with the following exceptions: Arg88, Ile152, Asp157, Trp158, Val162, Leu233, Lys240, Asp270, Arg296, Gly323, Cys326 and Ser338. As can be seen from Figure 3, especially in the inward-facing model of hFpn, most of the loss-of function mutations are located on the N-terminal half of the protein where Asp39, His43, and Asp181 are located. This region hosts also the residues building up Motif-A, essential for stabilizing the outward-facing conformation of the protein, allowing the release of the substrate. Indeed loss-of-function mutations affect residues Ala77, Gly80, Asp84 and Arg88 (Table 1). The latter three residues form hFpn Motif-A (Figure 2), and the mutations observed either do not allow the close contact between TM2 and TM11 needed to reach the outward-facing conformation (A77D, G80S/V, D84E) or disrupt the charge interaction between Asp84 and Arg88 needed to stabilize the same conformation (R88G/T). From this viewpoint, the outward-facing model of hFpn clearly shows that Asp157 is part of the charge network involving Asp84 and Arg88 (Figure 2) and indeed loss-of-function mutations affecting Asp157 are observed as well (D157G/Y/N; Table 1).Figure 3:Pathogenic mutations of hFpn mapped onto the structural models.Location of the residues affected by loss-of-function mutations (red) and gain-of-function mutations (green) in the hFpn inward-facing (A) and outward-facing (B) conformations.Concerning gain-of-function mutations, the outward-facing model provides a ready explanation of their effect. Indeed almost all of these mutations are clustered around an area centered on TM2 and TM11 where the hepcidin “docking” site (defined by Cys326 and His507 [28]) is located (Figure 3). These mutations involve residues Tyr64, Ala69, Ser71, Val72, Asn144, Cys326, Tyr501, Asp504 and His507 (Table 1), which define an almost continuous patch of the cavity of hFpn in the outward-facing conformation (Figure 4), in line with a homology model based on BdFpn in the outward open conformation [14]. Mutation of each of these residues likely modifies the hepcidin interaction surface on hFpn, resulting in a decreased or impaired binding of this hormone and failure to initiate hFpn internalization and degradation. This hypothesis is confirmed by docking simulations of hepcidin onto the outward-facing model of hFpn, carried out using ZDOCK (see Methods section for details). Indeed, in the best ranking complex, obtained by rescoring the docking poses with ZRANK, hepcidin nicely sits within the hFpn cavity, contacting all the residues building up the above mentioned surface patch (Figure 4). Interestingly, in this complex hepcidin N-terminal end contacts, almost all of the residues affected by gain-of-function mutations, in agreement with the experimental evidence that the first nine residues of hepcidin are critical for hFpn binding and for the disulphide exchange process with Cys326, which triggers hFpn internalization and degradation [31, 32]. Other residues involved in gain-of-function mutations are Gly204, Lys240 and Asp270. Gly204 is positioned at the extracellular end of TM6 near the membrane boundary and the short loop connecting TM5 and TM6, in a position in which the two α-helices are in close contact. Substitution of this residue with a bulkier residue probably does not affect hepcidin binding, but it may destabilize the outward-facing conformation and/or interfere with the internalization process. Lys240 is located in the long intracellular loop connecting TM6 and TM7, and it has been shown to be involved in hepcidin-dependent ubiquitination [33, 34]. Also, Asp270 is found in this loop and replacement with Val might modify the loop conformation and the accessibility of the Lys residues required for ubiquitination of hFpn.Figure 4:Structural details of the hFpn hepcidin binding site.(A) Residues affected by gain-of-function mutations (green) located in the hepcidin binding cavity mapped onto the molecular surface of hFpn in the outward-facing conformation. (B) Putative hFpn-hepcidin complex obtained by docking simulations. Note the close proximity between hepcidin N-terminal residues and hFpn residues affected by gain-of-function mutations.ConclusionIn this work, novel and improved structural models of the inward- and outward-facing conformations of hFpn, based on the recently solved BdFpn three-dimensional structures, have been presented. The models presented in this manuscript provide a novel insight into human Fpn structure-function relationships with respect to previously published models. In particular, the inward-facing model uncovers a structural role of the His43-Asp325 pair, never hypothesized before, also corroborated by compensative mutations observed in BdFpn in which an Asp28-His261 pair is observed; the outward-facing model highlights the presence also in hFpn of an amphipatic α-helix stabilizing this conformation, as observed in other members of the MFS, never postulated on the basis of previous models. The same model also provides the first view at atomic level of the Asp157 involvement in the Motif-A charge relay, this latter observation being particularly noteworthy as this residue is affected in loss-of-function mutants. Furthermore, the higher reliability of our outward-facing model allowed to obtain for the first time a structural model of the hFpn-hepcidin complex, which is fully in line with the current knowledge of hepcidin mechanism of action and consistent with the location of gain-of-function mutations. The models allowed the analysis at the structural level of the effect of the loss-of-function and gain-of-function mutations leading, respectively, to type 4A and type 4B hemochromatosis. In the absence of an experimental structure, the models here described represent a valuable alternative for structure-based functional studies on hFpn. Particularly interesting, in this respect, is the reliability of the outward-facing model as far as the hepcidin binding site is concerned. This model appears to be a good starting point for the design of hepcidin substitutes useful for the treatment of iron overload disorders.Author contributions: The authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.Research funding: None declared.Employment or leadership: None declared.Honorarium: None declared.Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.References1.Bogdan AR, Miyazawa M, Hashimoto K, Tsuji Y. Regulators of iron homeostasis: new players in metabolism, cell death, and disease. Trends Biochem Sci 2016;41:274–86.BogdanARMiyazawaMHashimotoKTsujiYRegulators of iron homeostasis: new players in metabolism, cell death, and diseaseTrends Biochem Sci201641274862.Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 2012;24:981–90.RayPDHuangBWTsujiYReactive oxygen species (ROS) homeostasis and redox regulation in cellular signalingCell Signal201224981903.Drakesmith H, Nemeth E, Ganz T. Ironing out ferroportin. Cell Metab 2015;22:777–87.DrakesmithHNemethEGanzTIroning out ferroportinCell Metab201522777874.Montosi G, Donovan A, Totaro A, Garuti C, Pignatti E, Cassanelli S, et al. Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene. J Clin Invest 2001;108:619–23.MontosiGDonovanATotaroAGarutiCPignattiECassanelliSAutosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) geneJ Clin Invest2001108619235.Pietrangelo A. The ferroportin disease: pathogenesis, diagnosis and treatment. Haematologica 2017;102:1972–84.PietrangeloAThe ferroportin disease: pathogenesis, diagnosis and treatmentHaematologica20171021972846.Yan N. Structural biology of the major facilitator superfamily transporters. Annu Rev Biophys 2015;44:257–83.7.YanNStructural biology of the major facilitator superfamily transportersAnnu Rev Biophys20154425783.77.Tortosa V, Bonaccorsi di Patti MC, Musci G, Polticelli F. The human iron exporter ferroportin. Insight into the transport mechanism by molecular modeling. Bio-Algorithms Med-Systems 2016;12:1–7.TortosaVBonaccorsi di PattiMCMusciGPolticelliFThe human iron exporter ferroportin. Insight into the transport mechanism by molecular modelingBio-Algorithms Med-Systems201612178.Wallace DF, Harris JM, Subramaniam VN. Functional analysis and theoretical modeling of ferroportin reveals clustering of mutations according to phenotype. Am J Physiol Cell Physiol 2010;298:C75–84.WallaceDFHarrisJMSubramaniamVNFunctional analysis and theoretical modeling of ferroportin reveals clustering of mutations according to phenotypeAm J Physiol Cell Physiol2010298C75849.Huang Y, Lemieux MJ, Song J, Auer M, Wang DN. Structure and mechanism of the glycerol-3 phosphate transporter from Escherichia coli. Science 2003;301:616–20.HuangYLemieuxMJSongJAuerMWangDNStructure and mechanism of the glycerol-3 phosphate transporter from Escherichia coliScience20033016162010.Le Gac G, Ka C, Joubrel R, Gourlaouen I, Lehn P, Mornon JP, et al. Structure-function analysis of the human ferroportin iron exporter (SLC40A1): effect of hemochromatosis type 4 disease mutations and identification of critical residues. Hum Mutat 2013;34:1371–80.Le GacGKaCJoubrelRGourlaouenILehnPMornonJPStructure-function analysis of the human ferroportin iron exporter (SLC40A1): effect of hemochromatosis type 4 disease mutations and identification of critical residuesHum Mutat20133413718011.Yin Y, He X, Szewczyk P, Nguyen T, Chang G. Structure of the multidrug transporter EmrD from Escherichia coli. Science 2006;312:741–4.YinYHeXSzewczykPNguyenTChangGStructure of the multidrug transporter EmrD from Escherichia coliScience2006312741412.Bonaccorsi di Patti MC, Polticelli F, Cece G, Cutone A, Felici F, Persichini T, et al. A structural model of human ferroportin and of its iron binding site. FEBS J 2014;281:2851–60.Bonaccorsi di PattiMCPolticelliFCeceGCutoneAFeliciFPersichiniTA structural model of human ferroportin and of its iron binding siteFEBS J201428128516013.Dang S, Sun L, Huang Y, Lu F, Liu Y, Gong H, et al. Structure of a fucose transporter in an outward-open conformation. Nature 2010;467:734–8.DangSSunLHuangYLuFLiuYGongHStructure of a fucose transporter in an outward-open conformationNature2010467734814.Taniguchi R, Kato HE, Deshpande CN, Wada M, Ito K, Ishitani R, et al. Outward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportin. Nat Commun 2015;6:8545.TaniguchiRKatoHEDeshpandeCNWadaMItoKIshitaniROutward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportinNat Commun20156854515.Bonaccorsi di Patti MC, Polticelli F, Tortosa V, Furbetta PA, Musci G. A bacterial homologue of the human iron exporter ferroportin. FEBS Lett 2015;21:3829–35.Bonaccorsi di PattiMCPolticelliFTortosaVFurbettaPAMusciGA bacterial homologue of the human iron exporter ferroportinFEBS Lett20152138293516.Fiser A, Do RK, Sali A. Modeling of loops in protein structures. Protein Sci 2000;9:1753–73.FiserADoRKSaliAModeling of loops in protein structuresProtein Sci2000917537317.Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006;22:195–201.ArnoldKBordoliLKoppJSchwedeTThe SWISS-MODEL Workspace: a web-based environment for protein structure homology modellingBioinformatics20062219520118.Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER suite: protein structure and function prediction. Nat Methods 2015;12:7–8.YangJYanRRoyAXuDPoissonJZhangYThe I-TASSER suite: protein structure and function predictionNat Methods2015127819.Wu S, Zhang Y. LOMETS: a local meta-threading-server for protein structure prediction. Nucleic Acids Res 2007;35:3375–82.WuSZhangYLOMETS: a local meta-threading-server for protein structure predictionNucleic Acids Res20073533758220.Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–402.AltschulSFMaddenTLSchäfferAAZhangJZhangZMillerWGapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Res199725338940221.Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 2010;38:W695–9.GoujonMMcWilliamHLiWValentinFSquizzatoSPaernJA new bioinformatics analysis tools framework at EMBL-EBINucleic Acids Res201038W695922.Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK – a program to check the stereochemical quality of protein structures. J App Cryst 1993;26:283–91.LaskowskiRAMacArthurMWMossDSThorntonJMPROCHECK – a program to check the stereochemical quality of protein structuresJ App Cryst1993262839123.Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 2014;42:W320–24.RobertXGouetPDeciphering key features in protein structures with the new ENDscript serverNucleic Acids Res201442W3202424.Jordan JB, Poppe L, Haniu M, Arvedson T, Syed R, Li V, et al. Hepcidin revisited, disulfide connectivity, dynamics, and structure. J Biol Chem 2009;284:24155–67.JordanJBPoppeLHaniuMArvedsonTSyedRLiVHepcidin revisited, disulfide connectivity, dynamics, and structureJ Biol Chem2009284241556725.Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng Z. ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics 2014;30:1771–3.PierceBGWieheKHwangHKimBHVrevenTWengZZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimersBioinformatics2014301771326.Pierce B, Weng Z. ZRANK: reranking protein docking predictions with an optimized energy function. Proteins 2007;67:1078–86.PierceBWengZZRANK: reranking protein docking predictions with an optimized energy functionProteins20076710788627.Mayr R, Janecke AR, Schranz M, Griffiths WJ, Vogel W, Pietrangelo A, et al. Ferroportin disease: a systematic meta-analysis of clinical and molecular findings. J Hepatol 2010;53:941–49.MayrRJaneckeARSchranzMGriffithsWJVogelWPietrangeloAFerroportin disease: a systematic meta-analysis of clinical and molecular findingsJ Hepatol2010539414928.Jiang D, Zhao Y, Wang X, Fan J, Heng J, Liu X, et al. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A. Proc Natl Acad Sci USA 2013;110:14664–9.JiangDZhaoYWangXFanJHengJLiuXStructure of the YajR transporter suggests a transport mechanism based on the conserved motif AProc Natl Acad Sci USA201311014664929.Madej MG, Soro SN, Kaback HR. Apo-intermediate in the transport cycle of lactose permease (LacY). Proc Natl Acad Sci USA 2012;109:E2970–8.MadejMGSoroSNKabackHRApo-intermediate in the transport cycle of lactose permease (LacY)Proc Natl Acad Sci USA2012109E2970830.Jiang D, Zhao Y, Wang X, Fan J, Henga J, Liua X, et al. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A. Proc Natl Acad Sci USA 2013;110:14664–69.JiangDZhaoYWangXFanJHengaJLiuaXStructure of the YajR transporter suggests a transport mechanism based on the conserved motif AProc Natl Acad Sci USA2013110146646931.Fernandes A, Preza GC, Phung Y, De Domenico I, Kaplan J, Ganz T, et al. The molecular basis of hepcidin-resistant hereditary hemochromatosis. Blood 2009;114:437–43.FernandesAPrezaGCPhungYDe DomenicoIKaplanJGanzTThe molecular basis of hepcidin-resistant hereditary hemochromatosisBlood20091144374332.Preza GC, Ruchala P, Pinon R, Ramos E, Qiao B, Peralta MA, et al. Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload. J Clin Investigation 2011;121:4880–8.PrezaGCRuchalaPPinonRRamosEQiaoBPeraltaMAMinihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overloadJ Clin Investigation20111214880833.Qiao B, Sugianto P, Fung E, del-Castillo-Rueda A, Moran-Jimenez MJ, Ganz T, et al. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination. Cell Met 2012;15:918–24.QiaoBSugiantoPFungEdel-Castillo-RuedaAMoran-JimenezMJGanzTHepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitinationCell Met2012159182434.Ross SL, Tran L, Winters A, Lee KJ, Plewa C, Foltz I, et al. Ferroportin internalization requires ferroportin lysines, not tyrosines or JAK-STAT. Cell Met 2012;15:905–17.RossSLTranLWintersALeeKJPlewaCFoltzIFerroportin internalization requires ferroportin lysines, not tyrosines or JAK-STATCell Met20121590517Supplemental Material:The online version of this article offers supplementary material (https://doi.org/10.1515/bams-2017-0029). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Bio-Algorithms and Med-Systems de Gruyter

An improved structural model of the human iron exporter ferroportin. Insight into the role of pathogenic mutations in hereditary hemochromatosis type 4

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©2017 Walter de Gruyter GmbH, Berlin/Boston
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10.1515/bams-2017-0029
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Abstract

IntroductionIron is the second most abundant metal on Earth’s crust and plays an essential biological role in all living organisms [1]. Biological properties of iron are linked to its efficient electron transfer properties, allowing it to accept or donate electrons. This ability enables it not only to participate as a catalyzing cofactor in various biochemical reactions but also to contribute to the formation of potentially damaging free radical species [2]. Thus, iron concentration in body tissues needs to be strongly regulated. In fact, it is well known that the imbalance of iron homeostasis has noticeable effects on human health [1].Ferroportin (Fpn) is a membrane protein representing the major cellular iron exporter, essential for metal translocation from cells into plasma [3]. It belongs to one of the largest secondary transporter families, the major facilitator superfamily (MFS), and together with hepcidin, a 25-amino-acid peptide that binds to hFpn and drives its degradation, it is a key player in the control of human iron homeostasis [3]. Mutations in the gene coding for hFpn cause type 4 hemochromatosis [4], an autosomal dominant iron overload condition. Mutations are generally classified into two subclasses: “loss-of-function” and “gain-of-function”. The majority of pathogenic variants are defined as “loss-of-function,” as they lead to decreased iron export from cells. These give rise to type 4A hemochromatosis, also known as Fpn disease [5]. On the other hand, “gain-of-function” mutations, responsible for type 4B hemochromatosis, confer resistance to the action of hepcidin. Hepcidin binding to Fpn leads to protein internalization, ubiquitination, and degradation. Gain-of-function mutations are named this way because they inhibit this mechanism and cause Fpn to permanently stay on the membrane and continue to be active in iron efflux.Although the importance of Fpn in human iron homeostasis has been widely demonstrated, its exact three-dimensional structure remains unknown. Based on the knowledge of the structure of other members of the MFS family, it was proposed that Fpn operates through an alternating-access mechanism that involves a movement of the two halves of the protein, together with local structural rearrangements. The rotation of the two domains composing the protein leads to three different conformational states: outward-facing, occluded, and inward-facing [6, 7]. Despite the lack of a crystal structure, different models of hFpn have been proposed. The first available hFpn model [8] can be considered a “low-resolution” one. In fact, the transmembrane helices were predicted and built de novo and then manually fitted over the three-dimensional structure of the Escherichia coli glycerol-3-phosphate transporter in the inward-facing conformation (PDB code: 1PW4 [9]). A few years later, Le Gac and coworkers [10] described a novel model obtained using the three-dimensional structure of the MFS transporter EmrD (PDB code: 2GFP [11]) in the occluded conformation. However, loops outside of the transmembrane helices were not modeled, and some critical structural details of this model were later shown to be incorrect. For instance, Asp181, a residue affected in loss-of-function mutants, was hypothesized to be exposed toward the membrane while it was later shown to be essential for iron binding and transport and thus exposed within the substrate-binding pocket [12]. The first structural models of hFpn in both the inward- and outward-facing conformations were later obtained by Bonaccorsi di Patti and coworkers [12] on the basis of the three-dimensional structures of the glycerol-3-phosphate transporter (PDB code:1PW4 [9]) and the FucP fucose transporter (PDB code: 3O7Q [13]), respectively. These models allowed to uncover and experimentally validate the identity of residues involved in iron binding and transport. Finally, a possible iron-transport mechanism was hypothesized on the basis of hFpn models in all three mechanistically relevant conformations (inward-facing, occluded, and outward-facing) [7].Recently, a bacterial homologue of hFpn has been identified in the predatory bacterium Bdellovibrio bacteriovorus [14, 15] and the three-dimensional structure of this protein (BdFpn) has been determined in the inward- and outward-facing conformational states. Taking advantage of the availability of these structures, the only ones available for a member of the Fpn family, updated models of hFpn in the inward- and outward-facing conformations have been built and a comprehensive structure-function analysis of known Fpn pathogenic mutations has been carried out.MethodsMolecular modelingThe structural models of hFpn have been built using the ab initio/threading approach implemented in the I-TASSER pipeline [5] and the homology modeling software packages MODELLER [16] and SwissModel [17]. In the case of I-TASSER and SwissModel, no query/template alignment has been provided in input. In fact, in the case of I-TASSER, the server uses LOMETS (Local Meta-Threading Server) to thread the query sequence through a representative PDB structure library to search for the possible folds compatible with the sequence of the protein of interest [18, 19]. SwissModel instead uses PSI-BLAST [20] to construct the best query-template alignment. In the case of MODELLER, a reliable sequence alignment between hFpn and BdFpn has been given in input. This was obtained using CLUSTAL-omega [21] to align the amino acid sequence (NCBI GI code reported within parentheses) of BdFpn (gi 426403971) to those of Fpn from Homo sapiens (gi 7657100), Bos taurus (gi 118151032), Rattus norvegicus (gi 158635998), Mus musculus (gi 7109247), Anas platyrhynchos (gi 483522646), Gallus (gi 61098366), Chelonia mydas (gi 465955074), Danio rerio (gi 7109245), Echinops telfairi (gi 507641023), Tetraodon nigroviridis (gi 47225539), Branchiostoma floridae (gi 229294181), Caenorhabditis elegans (gi 351061132) and Ciona intestinalis (gi 198437857). In all three cases, modeling has been carried out by specifying as templates the three-dimensional structures of BdFpn in the inward-facing (PDB code: 5AYO [14]) and outward-facing (PDB code: 5AYM [14]) conformation. The overall quality of the models has been assessed using PROCHECK [22], and the corresponding G-factor values (the PROCHECK G-factor threshold for good-quality three-dimensional structures being −0.5) are reported in Supplementary Materials Table S1.Structure-based sequence alignment of the models to the three-dimensional structure of BdFpn in the inward-facing and outward-facing conformations has been obtained using ESPript 3.0 [23].Docking simulationsDocking simulations between the three-dimensional structure of hepcidin (PDB code: 3H0T [24]) and the structural model of hFpn in the outward-facing conformation have been performed using the protein-protein docking server ZDOCK (version 3.0.2), which is based on a Fast Fourier transform algorithm and a scoring system combining shape complementarity, electrostatics and statistical potential terms [25]. The 2000 complexes predicted by ZDOCK were re-ranked using ZRANK, which uses a more detailed potential combining electrostatics, van der Waals and desolvation terms [26].Collection of pathogenic mutationsA list of all known hFpn pathogenic mutations has been obtained through a careful manual analysis of the available literature. Most mutations were retrieved from the comprehensive meta-analysis by Mayr et al. [27]; the list has recently been updated [5, 27].Results and discussionAnalysis of the structural models of hFpn in the inward- and outward-facing conformationsThe structural models of hFpn in the inward- and outward-facing conformations obtained with I-TASSER, MODELLER, and SwissModel are shown in Figure 1. To further assess the degree of agreement between the different models at the residue level, a structure-based sequence alignment has been carried out. As can be seen in Supplementary Materials Figures S1 and S2, the three different models display a high degree of consensus in the core structure (transmembrane helices) while, for obvious reasons, large deviations are observed at the level of the intracellular and extracellular surface loops. From this viewpoint, it must be noted that in the models obtained by homology modeling, the surface loops are not structured. On the contrary, the threading/ab initio approach implemented in I-TASSER, including replica exchange molecular dynamics simulations, allows to predict a putative structure of the large loops, which characterize hFpn. In other structurally characterized members of the MFS, these loops are involved in the inward to outward conformational change [28]. Therefore, once assessed, the substantial agreement between the three methods, the subsequent structural analyses were conducted on the hFpn models generated with I-TASSER.Figure 1:Schematic representation of the structural models of hFpn obtained in this study.Structural models of hFpn obtained with I-TASSER (tan), MODELLER (cyan), and SwissModel (purple) in the inward-facing (A) and outward-facing (B) conformations.The superposition of BdFpn structures with the hFpn models generated by I-TASSER yields RMSD values of 1.24 Å for the inward-facing state and of 1.06 Å for the outward-facing state, with the most noticeable differences being located at the level of the TM2-TM3 connecting loop. This difference is acceptable, as this loop is considerably longer in hFpn with respect to BdFpn, and has been subjected to an ab initio modeling procedure in the I-TASSER workflow.As already emerged from previous studies [7, 8, 10, 12, 14], the structural model of hFpn consists of 12 transmembrane helices (TM), which are arranged in two domains (N-domain and C-domain), each containing six consecutive transmembrane helices, surrounding a substrate translocation pore, with the N- and C-termini located on the intracellular side. Helices, 1, 2, 4, 5, 7, 8, 10 and 11 shape the central transport cavity, and helices 3, 6, 9 and 12 form the outer domain.Experimental studies carried out on hFpn have identified functional roles for selected residues in iron binding and transport. In agreement with the biochemical characterization of other MFS transporters, the hypothetical substrate-binding site of hFpn is located halfway into the membrane [6], with the TM7 contributing more than half of the interacting residues for ligand coordination. In detail, Asp39, Asp181 and Asp325, whose mutation completely abolishes the iron binding and transport ability [12], form a putative iron binding site (Figure 2); Arg466 mutation significantly reduces the iron transport ability of Fpn and has been hypothesized to act as an “electrostatic switch”, which, upon iron binding, facilitates the inward to outward transition of Fpn [7]. These residues are conserved in BdFpn, with the exception of hFpn Asp325, which substitutes His261 present in the orthologous position in BdFpn [15]. Unexpectedly, mutation of BdFpn His261 into Asp (mimicking hFpn in this regard) completely abolished iron transport [15]. Analysis of the latest inward-facing model of hFpn provides a likely explanation for this result. In fact, Asp325 is predicted to be located in the vicinity of His43 (Figure 2), and a compensative substitution of hFpn His43 with Asp28 in BdFpn is observed, indicating a functional role of an Asp-His pair (Asp325-His43 in hFpn; Asp28-His261 in BdFpn), which is disrupted in the BdFpn His261Asp mutant. Thus, the two Fpns contain similarly oriented His-Asp pairs, and this observation, together with the reduced iron transport ability observed upon mutation of Asp325 in hFpn [12] and His261 in BdFpn [15], indicates that this residues pair likely plays a role in iron binding and/or translocation. The overall structure of Fpn shows that the transporter consists of irregular, highly kinked transmembrane helices that could be stabilized by the hydrophobicity and/or size of the amino acid side chains. Several “loss-of-function” mutations are located in positions that could induce irregularities in helices [29] indicating that, although none of these residues is required for hFpn activity, the bulkyness of the side chain at these positions could be important for conformational flexibility.Figure 2:Selected, functionally-relevant structural details of the hFpn structural models.Schematic representation of (A) the hFpn putative iron binding site viewed from the cytoplasmic side in the inward-facing conformation; (B) Motif-A involving Gly80, Asp84, Arg88 and Asp157 in the outward-facing conformation (Gly80 position indicated by the grey ribbon color); (C) the cytoplasmic kinked, amphipatic α-helix interacting with the cytoplasmic ends of the transmembrane helices in the outward-facing conformation.Another important motif identified in this molecule is Motif-A [28] (Figure 2), located at the level of the cytoplasmic loop connecting TM2 and TM3, formed by residues Gly80 (Gly65 in BdFpn), Asp84 (Asp69 in BdFpn) and Arg88 (Arg73 in BdFpn) [7, 15]. This motif, largely conserved in members of the MFS family, does not likely play a direct role in the iron transport mechanism but may only function as a cytoplasmic anchor point probably coordinating the conformational changes of MFS transporters by stabilizing the outward-facing conformation [6, 30].An interesting feature of the outward-facing model of hFpn is the presence, on the cytoplasmic side, of a kinked amphipathic α-helix seating at the cytoplasm-membrane boundary and interacting with the cytoplasmic ends of the transmembrane α-helices (Figure 2). This helix corresponds to the long loop connecting the N- and C-terminal domains in the inward-facing conformation. Interestingly, in other members of the MFS, the structuring of this loop into an amphipathic α-helix has been suggested to drive the inward- to outward-facing conformation [30].Mapping of all known mutations on the structural models of hFpnTable 1 shows a list of all hFpn mutations known up to date, together with the corresponding clinical and biochemical phenotypes. From this viewpoint, it must be mentioned that a structure-function analysis of a restricted subset of these mutations was already reported by Le Gac and coworkers [10] on the basis of the hFpn structural model in the occluded conformation. However, as briefly mentioned in the Introduction, some of the structural details of this model were later shown not to be consistent with experimental data and, given the occluded conformation of the model, the role of mutations conferring hepcidin resistance was difficult to assess.Table 1:Classification of hFpn missense variants.MutationClinical phenotypeBiochemical phenotypeY64N/HNon classicalGain of functionA69TNon classicalGain of functionS71FNon classicalGain of functionV72FNon classicalGain of functionA77DVariableLoss of functionG80S/VVariableLoss of functionD84EClassicalLoss of functionR88G/TClassicalLoss of functionL129PClassicalLoss of functionN144H/D/TVariableGain of functionI152FClassicalLoss of functionD157G/Y/NClassicalLoss of functionW158L/CClassicalLoss of functionV162delClassicalLoss of functionL170FN/ALoss of functionN174IVariableLoss of functionR178QVariableN/AI180T–NeutralD181V/NClassicalLoss of functionQ182HClassicalLoss of functionN185DVariableLoss of functionG204S/RVariableGain of functionT230N–NeutralL233PVariableLoss of functionK240ENon classicalGain of functionQ248H–SNPM266T–NeutralG267DN/AN/AD270VNon classicalGain of functionR296QClassicalLoss of functionG323VN/ALoss of functionF324S–SNPC326S/Y/FNon classicalGain of functionS338RNon classicalGain of functionG339D–SNPL345F–NeutralI351V–NeutralR371Q–NeutralL384M/V–SNPF405S–SNPM432V–SNPP443L–SNPR489S/KVariableLoss of functionG490D/SVariableLoss of functionY501CNon classicalGain of functionD504NNon classicalGain of functionH507RNon classicalGain of functionR561G–SNPSNP, single nucleotide polymorphism; N/A, not available.Here we report a detailed analysis of the putative structural-functional role of the gain-of-function and loss-of-function hFpn mutations based on the novel models obtained. A large part of the mutations is located on transmembrane helices with the following exceptions: Arg88, Ile152, Asp157, Trp158, Val162, Leu233, Lys240, Asp270, Arg296, Gly323, Cys326 and Ser338. As can be seen from Figure 3, especially in the inward-facing model of hFpn, most of the loss-of function mutations are located on the N-terminal half of the protein where Asp39, His43, and Asp181 are located. This region hosts also the residues building up Motif-A, essential for stabilizing the outward-facing conformation of the protein, allowing the release of the substrate. Indeed loss-of-function mutations affect residues Ala77, Gly80, Asp84 and Arg88 (Table 1). The latter three residues form hFpn Motif-A (Figure 2), and the mutations observed either do not allow the close contact between TM2 and TM11 needed to reach the outward-facing conformation (A77D, G80S/V, D84E) or disrupt the charge interaction between Asp84 and Arg88 needed to stabilize the same conformation (R88G/T). From this viewpoint, the outward-facing model of hFpn clearly shows that Asp157 is part of the charge network involving Asp84 and Arg88 (Figure 2) and indeed loss-of-function mutations affecting Asp157 are observed as well (D157G/Y/N; Table 1).Figure 3:Pathogenic mutations of hFpn mapped onto the structural models.Location of the residues affected by loss-of-function mutations (red) and gain-of-function mutations (green) in the hFpn inward-facing (A) and outward-facing (B) conformations.Concerning gain-of-function mutations, the outward-facing model provides a ready explanation of their effect. Indeed almost all of these mutations are clustered around an area centered on TM2 and TM11 where the hepcidin “docking” site (defined by Cys326 and His507 [28]) is located (Figure 3). These mutations involve residues Tyr64, Ala69, Ser71, Val72, Asn144, Cys326, Tyr501, Asp504 and His507 (Table 1), which define an almost continuous patch of the cavity of hFpn in the outward-facing conformation (Figure 4), in line with a homology model based on BdFpn in the outward open conformation [14]. Mutation of each of these residues likely modifies the hepcidin interaction surface on hFpn, resulting in a decreased or impaired binding of this hormone and failure to initiate hFpn internalization and degradation. This hypothesis is confirmed by docking simulations of hepcidin onto the outward-facing model of hFpn, carried out using ZDOCK (see Methods section for details). Indeed, in the best ranking complex, obtained by rescoring the docking poses with ZRANK, hepcidin nicely sits within the hFpn cavity, contacting all the residues building up the above mentioned surface patch (Figure 4). Interestingly, in this complex hepcidin N-terminal end contacts, almost all of the residues affected by gain-of-function mutations, in agreement with the experimental evidence that the first nine residues of hepcidin are critical for hFpn binding and for the disulphide exchange process with Cys326, which triggers hFpn internalization and degradation [31, 32]. Other residues involved in gain-of-function mutations are Gly204, Lys240 and Asp270. Gly204 is positioned at the extracellular end of TM6 near the membrane boundary and the short loop connecting TM5 and TM6, in a position in which the two α-helices are in close contact. Substitution of this residue with a bulkier residue probably does not affect hepcidin binding, but it may destabilize the outward-facing conformation and/or interfere with the internalization process. Lys240 is located in the long intracellular loop connecting TM6 and TM7, and it has been shown to be involved in hepcidin-dependent ubiquitination [33, 34]. Also, Asp270 is found in this loop and replacement with Val might modify the loop conformation and the accessibility of the Lys residues required for ubiquitination of hFpn.Figure 4:Structural details of the hFpn hepcidin binding site.(A) Residues affected by gain-of-function mutations (green) located in the hepcidin binding cavity mapped onto the molecular surface of hFpn in the outward-facing conformation. (B) Putative hFpn-hepcidin complex obtained by docking simulations. Note the close proximity between hepcidin N-terminal residues and hFpn residues affected by gain-of-function mutations.ConclusionIn this work, novel and improved structural models of the inward- and outward-facing conformations of hFpn, based on the recently solved BdFpn three-dimensional structures, have been presented. The models presented in this manuscript provide a novel insight into human Fpn structure-function relationships with respect to previously published models. In particular, the inward-facing model uncovers a structural role of the His43-Asp325 pair, never hypothesized before, also corroborated by compensative mutations observed in BdFpn in which an Asp28-His261 pair is observed; the outward-facing model highlights the presence also in hFpn of an amphipatic α-helix stabilizing this conformation, as observed in other members of the MFS, never postulated on the basis of previous models. The same model also provides the first view at atomic level of the Asp157 involvement in the Motif-A charge relay, this latter observation being particularly noteworthy as this residue is affected in loss-of-function mutants. Furthermore, the higher reliability of our outward-facing model allowed to obtain for the first time a structural model of the hFpn-hepcidin complex, which is fully in line with the current knowledge of hepcidin mechanism of action and consistent with the location of gain-of-function mutations. The models allowed the analysis at the structural level of the effect of the loss-of-function and gain-of-function mutations leading, respectively, to type 4A and type 4B hemochromatosis. In the absence of an experimental structure, the models here described represent a valuable alternative for structure-based functional studies on hFpn. Particularly interesting, in this respect, is the reliability of the outward-facing model as far as the hepcidin binding site is concerned. This model appears to be a good starting point for the design of hepcidin substitutes useful for the treatment of iron overload disorders.Author contributions: The authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.Research funding: None declared.Employment or leadership: None declared.Honorarium: None declared.Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.References1.Bogdan AR, Miyazawa M, Hashimoto K, Tsuji Y. Regulators of iron homeostasis: new players in metabolism, cell death, and disease. Trends Biochem Sci 2016;41:274–86.BogdanARMiyazawaMHashimotoKTsujiYRegulators of iron homeostasis: new players in metabolism, cell death, and diseaseTrends Biochem Sci201641274862.Ray PD, Huang BW, Tsuji Y. Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling. Cell Signal 2012;24:981–90.RayPDHuangBWTsujiYReactive oxygen species (ROS) homeostasis and redox regulation in cellular signalingCell Signal201224981903.Drakesmith H, Nemeth E, Ganz T. Ironing out ferroportin. Cell Metab 2015;22:777–87.DrakesmithHNemethEGanzTIroning out ferroportinCell Metab201522777874.Montosi G, Donovan A, Totaro A, Garuti C, Pignatti E, Cassanelli S, et al. Autosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) gene. J Clin Invest 2001;108:619–23.MontosiGDonovanATotaroAGarutiCPignattiECassanelliSAutosomal-dominant hemochromatosis is associated with a mutation in the ferroportin (SLC11A3) geneJ Clin Invest2001108619235.Pietrangelo A. The ferroportin disease: pathogenesis, diagnosis and treatment. Haematologica 2017;102:1972–84.PietrangeloAThe ferroportin disease: pathogenesis, diagnosis and treatmentHaematologica20171021972846.Yan N. Structural biology of the major facilitator superfamily transporters. Annu Rev Biophys 2015;44:257–83.7.YanNStructural biology of the major facilitator superfamily transportersAnnu Rev Biophys20154425783.77.Tortosa V, Bonaccorsi di Patti MC, Musci G, Polticelli F. The human iron exporter ferroportin. Insight into the transport mechanism by molecular modeling. Bio-Algorithms Med-Systems 2016;12:1–7.TortosaVBonaccorsi di PattiMCMusciGPolticelliFThe human iron exporter ferroportin. Insight into the transport mechanism by molecular modelingBio-Algorithms Med-Systems201612178.Wallace DF, Harris JM, Subramaniam VN. Functional analysis and theoretical modeling of ferroportin reveals clustering of mutations according to phenotype. Am J Physiol Cell Physiol 2010;298:C75–84.WallaceDFHarrisJMSubramaniamVNFunctional analysis and theoretical modeling of ferroportin reveals clustering of mutations according to phenotypeAm J Physiol Cell Physiol2010298C75849.Huang Y, Lemieux MJ, Song J, Auer M, Wang DN. Structure and mechanism of the glycerol-3 phosphate transporter from Escherichia coli. Science 2003;301:616–20.HuangYLemieuxMJSongJAuerMWangDNStructure and mechanism of the glycerol-3 phosphate transporter from Escherichia coliScience20033016162010.Le Gac G, Ka C, Joubrel R, Gourlaouen I, Lehn P, Mornon JP, et al. Structure-function analysis of the human ferroportin iron exporter (SLC40A1): effect of hemochromatosis type 4 disease mutations and identification of critical residues. Hum Mutat 2013;34:1371–80.Le GacGKaCJoubrelRGourlaouenILehnPMornonJPStructure-function analysis of the human ferroportin iron exporter (SLC40A1): effect of hemochromatosis type 4 disease mutations and identification of critical residuesHum Mutat20133413718011.Yin Y, He X, Szewczyk P, Nguyen T, Chang G. Structure of the multidrug transporter EmrD from Escherichia coli. Science 2006;312:741–4.YinYHeXSzewczykPNguyenTChangGStructure of the multidrug transporter EmrD from Escherichia coliScience2006312741412.Bonaccorsi di Patti MC, Polticelli F, Cece G, Cutone A, Felici F, Persichini T, et al. A structural model of human ferroportin and of its iron binding site. FEBS J 2014;281:2851–60.Bonaccorsi di PattiMCPolticelliFCeceGCutoneAFeliciFPersichiniTA structural model of human ferroportin and of its iron binding siteFEBS J201428128516013.Dang S, Sun L, Huang Y, Lu F, Liu Y, Gong H, et al. Structure of a fucose transporter in an outward-open conformation. Nature 2010;467:734–8.DangSSunLHuangYLuFLiuYGongHStructure of a fucose transporter in an outward-open conformationNature2010467734814.Taniguchi R, Kato HE, Deshpande CN, Wada M, Ito K, Ishitani R, et al. Outward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportin. Nat Commun 2015;6:8545.TaniguchiRKatoHEDeshpandeCNWadaMItoKIshitaniROutward- and inward-facing structures of a putative bacterial transition-metal transporter with homology to ferroportinNat Commun20156854515.Bonaccorsi di Patti MC, Polticelli F, Tortosa V, Furbetta PA, Musci G. A bacterial homologue of the human iron exporter ferroportin. FEBS Lett 2015;21:3829–35.Bonaccorsi di PattiMCPolticelliFTortosaVFurbettaPAMusciGA bacterial homologue of the human iron exporter ferroportinFEBS Lett20152138293516.Fiser A, Do RK, Sali A. Modeling of loops in protein structures. Protein Sci 2000;9:1753–73.FiserADoRKSaliAModeling of loops in protein structuresProtein Sci2000917537317.Arnold K, Bordoli L, Kopp J, Schwede T. The SWISS-MODEL Workspace: a web-based environment for protein structure homology modelling. Bioinformatics 2006;22:195–201.ArnoldKBordoliLKoppJSchwedeTThe SWISS-MODEL Workspace: a web-based environment for protein structure homology modellingBioinformatics20062219520118.Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y. The I-TASSER suite: protein structure and function prediction. Nat Methods 2015;12:7–8.YangJYanRRoyAXuDPoissonJZhangYThe I-TASSER suite: protein structure and function predictionNat Methods2015127819.Wu S, Zhang Y. LOMETS: a local meta-threading-server for protein structure prediction. Nucleic Acids Res 2007;35:3375–82.WuSZhangYLOMETS: a local meta-threading-server for protein structure predictionNucleic Acids Res20073533758220.Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–402.AltschulSFMaddenTLSchäfferAAZhangJZhangZMillerWGapped BLAST and PSI-BLAST: a new generation of protein database search programsNucleic Acids Res199725338940221.Goujon M, McWilliam H, Li W, Valentin F, Squizzato S, Paern J, et al. A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res 2010;38:W695–9.GoujonMMcWilliamHLiWValentinFSquizzatoSPaernJA new bioinformatics analysis tools framework at EMBL-EBINucleic Acids Res201038W695922.Laskowski RA, MacArthur MW, Moss DS, Thornton JM. PROCHECK – a program to check the stereochemical quality of protein structures. J App Cryst 1993;26:283–91.LaskowskiRAMacArthurMWMossDSThorntonJMPROCHECK – a program to check the stereochemical quality of protein structuresJ App Cryst1993262839123.Robert X, Gouet P. Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res 2014;42:W320–24.RobertXGouetPDeciphering key features in protein structures with the new ENDscript serverNucleic Acids Res201442W3202424.Jordan JB, Poppe L, Haniu M, Arvedson T, Syed R, Li V, et al. Hepcidin revisited, disulfide connectivity, dynamics, and structure. J Biol Chem 2009;284:24155–67.JordanJBPoppeLHaniuMArvedsonTSyedRLiVHepcidin revisited, disulfide connectivity, dynamics, and structureJ Biol Chem2009284241556725.Pierce BG, Wiehe K, Hwang H, Kim BH, Vreven T, Weng Z. ZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimers. Bioinformatics 2014;30:1771–3.PierceBGWieheKHwangHKimBHVrevenTWengZZDOCK server: interactive docking prediction of protein–protein complexes and symmetric multimersBioinformatics2014301771326.Pierce B, Weng Z. ZRANK: reranking protein docking predictions with an optimized energy function. Proteins 2007;67:1078–86.PierceBWengZZRANK: reranking protein docking predictions with an optimized energy functionProteins20076710788627.Mayr R, Janecke AR, Schranz M, Griffiths WJ, Vogel W, Pietrangelo A, et al. Ferroportin disease: a systematic meta-analysis of clinical and molecular findings. J Hepatol 2010;53:941–49.MayrRJaneckeARSchranzMGriffithsWJVogelWPietrangeloAFerroportin disease: a systematic meta-analysis of clinical and molecular findingsJ Hepatol2010539414928.Jiang D, Zhao Y, Wang X, Fan J, Heng J, Liu X, et al. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A. Proc Natl Acad Sci USA 2013;110:14664–9.JiangDZhaoYWangXFanJHengJLiuXStructure of the YajR transporter suggests a transport mechanism based on the conserved motif AProc Natl Acad Sci USA201311014664929.Madej MG, Soro SN, Kaback HR. Apo-intermediate in the transport cycle of lactose permease (LacY). Proc Natl Acad Sci USA 2012;109:E2970–8.MadejMGSoroSNKabackHRApo-intermediate in the transport cycle of lactose permease (LacY)Proc Natl Acad Sci USA2012109E2970830.Jiang D, Zhao Y, Wang X, Fan J, Henga J, Liua X, et al. Structure of the YajR transporter suggests a transport mechanism based on the conserved motif A. Proc Natl Acad Sci USA 2013;110:14664–69.JiangDZhaoYWangXFanJHengaJLiuaXStructure of the YajR transporter suggests a transport mechanism based on the conserved motif AProc Natl Acad Sci USA2013110146646931.Fernandes A, Preza GC, Phung Y, De Domenico I, Kaplan J, Ganz T, et al. The molecular basis of hepcidin-resistant hereditary hemochromatosis. Blood 2009;114:437–43.FernandesAPrezaGCPhungYDe DomenicoIKaplanJGanzTThe molecular basis of hepcidin-resistant hereditary hemochromatosisBlood20091144374332.Preza GC, Ruchala P, Pinon R, Ramos E, Qiao B, Peralta MA, et al. Minihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overload. J Clin Investigation 2011;121:4880–8.PrezaGCRuchalaPPinonRRamosEQiaoBPeraltaMAMinihepcidins are rationally designed small peptides that mimic hepcidin activity in mice and may be useful for the treatment of iron overloadJ Clin Investigation20111214880833.Qiao B, Sugianto P, Fung E, del-Castillo-Rueda A, Moran-Jimenez MJ, Ganz T, et al. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination. Cell Met 2012;15:918–24.QiaoBSugiantoPFungEdel-Castillo-RuedaAMoran-JimenezMJGanzTHepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitinationCell Met2012159182434.Ross SL, Tran L, Winters A, Lee KJ, Plewa C, Foltz I, et al. Ferroportin internalization requires ferroportin lysines, not tyrosines or JAK-STAT. Cell Met 2012;15:905–17.RossSLTranLWintersALeeKJPlewaCFoltzIFerroportin internalization requires ferroportin lysines, not tyrosines or JAK-STATCell Met20121590517Supplemental Material:The online version of this article offers supplementary material (https://doi.org/10.1515/bams-2017-0029).

Journal

Bio-Algorithms and Med-Systemsde Gruyter

Published: Dec 20, 2017

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