Bio-Algorithms and Med-Systems 2016; 12(1): 1–7

Valentina Tortosa, Maria Carmela Bonaccorsi di Patti, Giovanni Musci and Fabio Polticelli* The human exporter ferroportin. Insight into the transport mechanism by molecular modeling

DOI 10.1515/bams-2015-0034 system [1]. The synthesis of Fpn is regulated via the Received September 25, 2015; accepted November 4, 2015; previously ­iron-responsive element/iron regulatory system published online December 11, 2015 at the cellular level [2] and by at the organ- ism level [3, 4]. Hepcidin is a small peptide that plays a Abstract: Ferroportin, a membrane belonging to key role in the regulation of iron homeostasis by virtue the major facilitator superfamily of transporters, is the of its ability to bind Fpn, and induce its internalization only vertebrate iron exporter known so far. Several ferro- and subsequent degradation [3, 4]. The final effect is the portin mutations lead to the so-called ferroportin disease inhibition of iron efflux from duodenal , mac- or type 4 hemochromatosis, characterized by two distinct rophages, and into the bloodstream [5, 6]. iron accumulation phenotypes depending on whether the Hepcidin expression is probably transcriptionally regu- mutation affects the activity of the protein or its degra- lated, although the exact mechanisms are not completely dation pathway. Through extensive molecular modeling defined [4, 7, 8]. The hepcidin–Fpn axis is the principal analyses using the structure of all known major facilita- regulator of extracellular iron homeostasis in health and tor superfamily members as templates, multiple struc- disease [5]. tural models of ferroportin in the three mechanistically In humans, Fpn mutations are associated with type relevant conformations (inward open, occluded, and out- IV hemochromatosis or “ferroportin disease.” Most muta- ward open) have been obtained. The best models, selected tions are “loss of function” as they affect plasma mem- on the ground of experimental data available on wild-type brane localization or iron export ability, and lead to a and mutant ferroportion, provide for the first time a pre- phenotype characterized by a high serum concen- diction at the atomic level of the dynamics of the trans- tration, relative plasma iron deficiency, and preferential porter. Based on these results, a possible mechanism for iron retention in reticuloendothelial cells [9–12]. On the iron export is proposed. other hand, some “gain-of-function” mutations do not Keywords: ferroportin; iron transport; major facilitator impair the function of Fpn but rather result in hepcidin superfamily; molecular modeling. resistance and in the anomalous retention of the active protein on the membrane. The pathological signa- tures are in this case different, with a high saturation and parenchymal [9, 10, 12–15]. Introduction The topology of Fpn has not been exactly defined; in fact, no experimental data are available up to now on Ferroportin (Fpn), the only known mammalian iron Fpn three-dimensional structure. Structural bioinformat- exporter, is essential for transporting iron across the baso- ics analyses of Fpn revealed that the protein conforms lateral surface of enterocytes into the blood circulation to the general plan of the major facilitator superfamily system and for recycling iron in the reticuloendothelial (MFS) transporters [16]. This superfamily represents the largest group of secondary active membrane transport- *Corresponding author: Fabio Polticelli, Department of Sciences, ers and plays an important role in multiple physiologi- Roma Tre University, Viale Guglielmo Marconi 446, 00146 Rome, cal processes [17–19]. A canonical MFS fold comprises Italy, Phone: +39-06-57336362, Fax: +39-06-57336321, E-mail: [email protected]. 12 transmembrane helices (TMs) that are organized into http://orcid.org/0000-0002-7657-2019; and National Institute two folded domains, an N- and a C-terminal domain, of Nuclear Physics, Roma Tre Section, Rome, Italy each containing six consecutive TMs [20]. In turn, these Valentina Tortosa: Department of Sciences, Roma Tre University, two domains display a twofold pseudosymmetry related Rome, Italy by an axis perpendicular to the membrane plane [20]. Maria Carmela Bonaccorsi di Patti: Department of Biochemical Sciences, Sapienza University of Roma, Rome, Italy The rotation­ of one domain relative to the other allows Giovanni Musci: Department Biosciences and Territory, University of the conformational changes necessary for the “alternate Molise, Pesche, Italy access mechanism” of transport. This is achieved through 2 Tortosa et al.: Ferroportin iron transport mechanism cycling from an “inward-open” to an “outward-open” energy, the global topology, and the hydrogen-bonding ­conformation through an intermediated “occluded” con- network of the model [28]. formation and vice versa [20]. In this work, I-TASSER was used specifying as tem- Recently, two structural models of human Fpn have plates all the known three-dimensional structures of the been obtained by independent groups [10, 21]. The first MFS transporters (22 structures; see Table 1). However, one, built by modeling based on another threading-based LOMETS restraints were also used with member of the MFS, corresponds to Fpn structure in the the purpose of modeling the unaligned regions as well as “occluded” state and allowed to prove the crucial role adjusting the reassembly of aligned regions. The overall of Trp42 in both the iron transport mechanism and the quality of the 22 models generated by I-TASSER has been binding of hepcidin [10]. Most recently, a second model of evaluated using PROCHECK [50] and the model quality Fpn in the inward-open conformation has been obtained parameter C-score provided in the I-TASSER output (Sup- by ab initio modeling [21]. Analysis of this model allowed plementary Materials Table S1). Further, to choose the best identifying a potential iron binding site, centered around Fpn model in each of the functionally relevant conforma- residues Asp39 and Asp181, whose importance was con- tions, the 16 models displaying a G-factor value greater firmed through site-directed mutagenesis [21]. In the same than –0.5 (the PROCHECK G-factor threshold for good study, an essential role in iron transport was also estab- quality three dimensional structures) have been analyzed lished for Asp325 and Arg466, although their involvement according to criteria derived from available experimental in the transport mechanism remained undefined [21]. data that are described in detail in the Results section. In this work, we describe new structural models of Fpn obtained in the three mechanistically relevant con- formations (inward open, occluded, and outward open). Iron binding site detection Fpn structure was modeled using as templates all the known structures of MFS members. Twenty-two different Prediction of the presence of iron binding sites has been models have been obtained and analyzed in the context of carried out using the software LIBRA (Ligand Binding Site known experimental data in order to select the best model for each conformation. The three models selected provide Table 1: Known MSF transporters’ three-dimensional structures a prediction at the atomic level of the conformational used as templates to build Fpn models in the three mechanistically changes occurring in Fpn during iron transport and allow relevant conformational states. proposing a possible mechanism for iron export. Template structure PDB code Conformation Refs.

GlcP 4LDS Inward [29] GlpT 1PW4 Inward [30] Materials and methods GLUT-1 4PYP Inward [31] LacY 1PV7 Inward [32] NRT1.1 4OH3 Inward [33] Molecular modeling PepTSt (POT-family) 4APS Inward [34]

PepTSo (POT-family) 4TPH Inward [35] The structural models of Fpn were built using the ab initio/ XylE 4QIQ Inward [36] threading approach implemented in the I-TASSER pipe- YbgH (POT-family) 4Q65 Inward [37] NarK 4JR9 Partially inward [38] line [22–24]. NarU 4IU9 Partially inward [39] This consists of four general steps. In the first step, POT (POT-family) 4IKV Partially inward [40] the query sequence is threaded by LOMETS (Local Meta- NarU 4IU9 Inward partially occluded [39] Threading Server) through a representative Protein Data XylE 4JA3 Inward partially occluded [41] Bank (PDB) structure library to search for the possible PipT 4JO5 Inward occluded [42] folds compatible with the sequence of the protein of inter- EmrD 2GFP Occluded [43] PepT (POT-family) 2XUT Occluded [44] est [22, 25]. In the second step, the sequence is divided So XylE 4GBY Occluded [45] into threading-aligned and threading-unaligned regions. Lacy 4OAA Outward partially occluded [46] Fragments derived from the threading-aligned regions MelB 4M64 Outward partially occluded [47] are reassembled by replica-exchange Monte Carlo simu- FucP 3O7Q Outward [48] lations [26], while the structure of unaligned regions is YajR 3WDO Outward [49] built by ab initio folding simulations [27]. Models are then Note how no single transporter has been crystallized in all the three refined in an iterative procedure that optimizes the free conformations. Tortosa et al.: Ferroportin iron transport mechanism 3

Recognition Application) in conjunction with a database indicated that Motif A is essential to stabilize the outward- of known iron binding sites derived from the PDB. LIBRA open conformation through formation of a salt bridge is based on a graph theory approach to find the largest between the Asp and Arg residues of the motif (see Ref. subset of similar residues between an input protein and a [54] and references therein). The Motif A sequence signa- collection of known binding sites [51]. ture is conserved in Fpn and is formed by residues Gly80, Asp84, and Arg88. Thus, an additional criterion for Fpn best models selection has been the formation of a salt bridge between Asp84 and Arg88 in the outward-open Results conformation. The three models that better conformed to these cri- Using the template structures listed in Table 1, 22 different teria were those built using as templates the Escherichia molecular models of Fpn were initially generated (Sup- coli glycerol-3- transporter GlpT (Ref. [30]; PDB plementary Material, Figure S1). All the models display code 1PW4) for the inward-open conformation, the E. coli the typical fold of MFS proteins with 12 TMs spanning symporter proton:xylose XylE (Ref. [45]; PDB code 4GBY) the membrane and the N- and C-termini located on the for the occluded conformation, and the E. coli fucose intracellular side. For most of the models obtained, the transporter FucP (Ref. [48]; PDB code 3O7Q) for the out- overall quality is fairly good, as judged by PROCHECK ward-open conformation. analysis and C-score values (see Supplementary Material, Figure 1 shows a schematic representation of the Table S1). In fact, in 18 cases out of 22, the percentage of three models, which provide a prediction at an atomic residues in the allowed regions of the Ramachandran plot level of the conformational changes occurring in Fpn is > 97% and in 16 cases out of 22 the overall G-factor cal- during iron transport. In the inward-open conformation, culated by PROCHECK is ≥ –0.5, the threshold for high- the protein displays a wide channel accessible from the quality three-dimensional structures [50]. Further, in 15 intracellular side, which closes approximately in the cases out of 22, the C-score is greater than –2.5, the typical middle of the membrane plane at the level of residues range for molecular models being –5 to 2 [22]. Asp39 and Asp181. Interestingly, in this conformation, In order to select the three best models representative Asp325 forms a salt bridge with Arg466 with its car- of the inward-open, occluded, and outward-open confor- boxyl group facing away from the Asp39-Asp181 couple mation of Fpn, the 16 models with G-factor ≥ –0.5 were (Figure 2A). In the occluded conformation, the salt further screened according to the following experimen- bridge is broken and Asp325 moves toward Asp39 and tally derived considerations: Asp181, forming a site characterized by a high density Asp39, Asp181, and Asp325, whose mutation com- of negative charge (Figure 2B). Indeed, ligand binding pletely abolishes the iron binding and transport ability sites detection, using the LIBRA application in conjunc- of Fpn, form a putative iron binding site in the inward- tion with a database of structurally characterized iron open and occluded conformations. In a good model, these binding sites (Ref. [51]; see Materials and methods for residues should be placed at a relative distance < 14 Å, as details), reveals that the substructure formed by His32, observed in the structurally characterized iron binding Asp39, Asp181, and Asn185 displays a stereochemistry sites [21]. very similar to that of the Fe(II) binding site of the metal Arg466 mutation significantly reduces the iron trans- transporter MntR [55] (Supplementary Material, Figure port ability of Fpn and has been hypothesized to act as S2). In the outward-open model of Fpn, Asp39, Asp181, an “electrostatic switch,” which, upon iron binding, and Asp325 move away from each other, in agreement facilitates the inward to outward transition of Fpn [21]. with the notion that when the protein takes up this con- To establish electrostatic interactions with iron, this formation, the metal is released (Figure 2C). residue should be located at a distance < 10 Å (the typical In the outward-open model, it is worthwhile to Debye length in biological systems [52]), from the puta- mention the presence of the canonical Motif A [Gly 80]+1, tive iron binding site in the inward-open and occluded [Asp 84]+5, [Arg 88]+9, in which Gly80 allows tight packing conformations. of TM2 and TM11 and Asp84 establishes electrostatic Most members of the MFS display a conserved interactions with the N-terminal end of TM11 (backbone sequence signature [Gly]+1 [Asp]+5 [Arg]+9, located at the nitrogen of Ile491) and Arg88 (Figure 3). These interac- level of the cytoplasmic loop connecting TM2 and TM3, tions are predicted to stabilize the outward-open con- which has been named Motif A [53]. The crystal struc- formation of Fpn, as observed for other members of the ture of the MSF transporter YajR and mutational analyses MFS. 4 Tortosa et al.: Ferroportin iron transport mechanism

Figure 2: Schematic representation of the Fpn putative iron binding site in the inward-open (top panel), occluded (center panel), and outward-open (bottom panel) conformations.

Figure 1: Schematic representation of the structural models of Fpn in the inward-open (A), occluded (B), and outward-open (C) conformations.

Discussion

The analysis of the Fpn models obtained in the three dif- ferent functional conformations allows to rationalize the Figure 3: Schematic representation of Motif A in the outward-open available experimental data and to formulate hypotheses model of Fpn. Tortosa et al.: Ferroportin iron transport mechanism 5 on the role of specific residues in iron binding and trans- and Arg269 on TM7), which in the glycerol-3-­phosphate location. It is worth remarking that, in the inward- GlpT are thought to drive the motion of TM1 open conformation, Asp325 is involved in a salt bridge toward TM7, and the transition from the inward-open with Arg466 with its carboxyl group facing away from to the occluded state, upon binding of the negatively the Asp39-Asp181 couple. Thus, in this conformation, charged glycerol-3-phosphate ligand [30]. binding of Fe(II) probably involves only the latter couple Regarding the outward-open model, it is worthwhile of residues. In the transition from the inward open to discussing the presence of Motif A, formed by residues the occluded conformation, Asp325 moves away from [Gly 80]+1, [Asp 84]+5, and [Arg 88]+9, which is essential to Arg466 and toward the pair Asp39-Asp181. For the intrin- coordinate the conformational changes of MSF transport- sic methodological limits of modeling, the model has ers [54]. The presence of this motif in Fpn helps to explain been obtained in the absence of iron and thus cannot the association of mutations G80S and R88G with type IV fully represent the Fpn occluded state. However, the hemochromatosis [58]. In fact, the outward-open model high negative charge density region observed in this con- of Fpn indicates that mutation of Gly80 would impair formation appears well fit to host an iron binding site. the correct interdomain helix packing, while mutation of This hypothesis is in agreement with the experimental Arg88 would destabilize the outward-open conformation observation that mutations D39A, D181V, and D325A by disruption of the salt bridge with Asp84. lead to significant impairment of both iron binding and In conclusion, the Fpn models described in the iron efflux in cells transfected with the mutated Fpn present work, together with available experimental data [21]. Moreover, the mutation D181V has been found in on wild-type and mutant Fpn, allow hypothesizing the members of Italian families affected by type 4 hemo- following mechanism of iron transport. In the inward- chromatosis [56], further confirming the crucial role of open conformation, iron would bind to the Asp39-Asp181 Asp181. The occluded Fpn model also supports the role couple, His32, and Asn185, possibly contributing to metal of Arg466 as an electrostatic switch. In fact, while in the chelation. Iron binding would attract toward the metal inward-open conformation, the arginine charge is stabi- coordination sphere Asp325, driving the motion of TM1 lized by Asp325; in the occluded conformation, Arg466 toward TM7 and the transition to the occluded state. At has no charge partner and therefore is in a high energy the same time, rupture of the Asp325-Arg466 salt bridge state from an electrostatic viewpoint, being located and repulsion of Arg466 would occur, which would gen- in the partially desolvated protein interior. This high erate a high energy state due to the partially desolvated energy state could trigger the conformational change to arginine charge. This high energy state would be relaxed the outward-open conformation in which, according to upon transition to the outward-open state, facilitated by the corresponding Fpn model, Arg466 is fully solvent the electrostatic interactions formed within the Motif A. At accessible. It must be considered that, in the presence of this stage, iron ligands would move apart from each other iron, the rupture of the Asp325-Arg466 salt bridge would and the metal released in the extracellular space. be facilitated by electrostatic attraction of Asp325 toward At present, the available data do not allow to under- the metal bound by the Asp39-Asp181 couple and elec- stand if a coupling mechanism for iron transport (symport trostatic repulsion between the metal and the Arg466 or antiport) is at play in Fpn or iron is simply transported guanidinium group. From this viewpoint, a very interest- following its concentration gradient. However, the puta- ing observation is the stereochemical similarity detected tive transport mechanism outlined above will be useful between Fpn residues His32, Asp39, Asp181, and Asn185, to inspire additional mutagenesis studies that can help and the Fe(II) binding site of the metal transporter MntR clarify these aspects. [55] (Supplementary Materials Figure S2). Interestingly, mutation of His32 in rat Fpn and mutation of Asn185 in Author contributions: All the authors have accepted human Fpn both lead to a nonfunctional protein [49, responsibility for the entire content of this submitted 57]. It appears thus likely that the site formed by His32, manuscript and approved submission. Asp39, Asp181, and Asn185 is the initial Fe(II) binding Research funding: None declared. site whose coordination sphere would be completed by Employment or leadership: None declared. Asp325, driving the motion of TM1 toward TM7 and the Honorarium: None declared. transition from the inward open to the occluded state. Competing interests: The funding organization(s) played This hypothesis is further supported by the fact that no role in the study design; in the collection, analysis, and Asp39 and Asp325 are located in orthologous positions interpretation of data; in the writing of the report; or in the on TM1 and TM7 of two arginine residues (Arg45 on TM1 decision to submit the report for publication. 6 Tortosa et al.: Ferroportin iron transport mechanism

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