Phosphate is crucial for structural and metabolic needs including nucleotide and lipid synthesis, signalling, and chemical energy storage. Essential for phosphate uptake in plants and fungi are proton-coupled transporters of the Major Facilitator Super-family (MFS), which also have a function in sensing external phosphate levels as transceptors1–5. Here we report the 2.9 Å structure of a fungal high affinity phosphate importer, PiPT, in an inward-facing occluded state, with bound phosphate visible in the membrane buried binding site. The structure indicates both proton and phosphate exit pathways and suggests a modified asymmetrical 'Rocker-Switch' mechanism of phosphate transport. PiPT is related to several human transporter families, most notably the organic cation and anion transporters of the Solute Carrier Family (SLC22), which are implicated in cancer-drug resistance6,7. We modelled representative cation and anion SLC22 transporters based on the PiPT structure to surmise the structural basis for substrate binding and charge selectivity in this important family. The PiPT structure demonstrates and expands on principles of substrate transport by the MFS transporters and illuminates principles of phosphate uptake in particular.
The Major Facilitator Super-family is the largest super-family of secondary active transporters and its diverse members generally function as symporters or antiporters driven by proton or sodium gradients1. Structures of eight bacterial MFS transporters have been determined by 2D and 3D crystallography8–15. Based on the first of these a 'Rocker-Switch' mechanism was proposed9,10, suggesting that the symmetry related N- and C-domains rock back and forth as 'banana-shaped' rigid bodies with the central substrate binding site as the pivot point. However, structures of other MFS transporters in the occluded state adopt a compact arrangement of helices around the substrate binding site8,11,13 and a similarly occluded and compact structure for the Lactose Permease (LacY) has been suggested by molecular dynamics simulations16, double electron-electron resonance measurements17 and homology modeling18. This indicates that rigid body movements alone are not sufficient to explain translocation in the MFS super-family.
Piriformospora indica is an endophytic fungus that colonizes roots of many plant species and promotes growth19. We have recently shown the P. indica Phosphate Transporter (PiPT) to be a high affinity phosphate transporter involved in improving phosphate nutrition-levels in the host-plant20. PiPT belongs to the Phosphate:H+ Symporter (PHS) family within the Major Facilitator Super-family1. It is highly homologous to the Saccharomyces cerevisiae high affinity phosphate transporter, Pho84, and to plant phosphate transporters (Supplementary Fig. 1, Supplementary Table 1). It also shares homology with the human Solute Carrier alpha-group (SLC-α), especially the SLC22 family of human organic anion and cation transports, the SLC2 family of glucose facilitative transporters (GLUTs), and the related Synaptic Vesicle 2 Protein family (Supplementary Table 1)7,21,22.
The structure of PiPT in complex with its substrate, inorganic phosphate, was determined to 2.9 Å resolution by experimental phasing (Fig. 1) and refined to a free crystallographic R-factor of 25.9% (Supplementary Figs. 2, 3 and 4, Supplementary Table 2). PiPT confirms that the MFS-fold found in bacteria is conserved in eukaryotes. PiPT has 12 transmembrane helices (M1-M12) divided into two homologous domains (N- and C-domain) related by a quasi-twofold symmetry perpendicular to the membrane plane. The structure includes residues 30 to 518 except for 67 residues in the flexible linker between N- and C-domain, predicted from sequence to be disordered. This disordered linker region in PiPT contains no discernible structure in the solved state of the protein, as seen in several other MFS structures10,13,14. The linker has no sequence similarity to the four-helix bundle domain observed in the bacterial GLUT homologue XylE15.
The overall conformation of PiPT is similar to structures of MFS transporters solved in the occluded state8,11,13 with the two domains forming a clam-shell like arrangement around a central membrane-buried binding site where the phosphate is bound. To the extracellular side of the binding site a cluster of 3 phenyl residues (F50, F327, F369) (Fig. 1a) block the entry pathway, and the distance from the phosphate site to the extracellular solvent is ~20 Å. The intracellular side of the binding site is also occluded but less so. The helix M4 blocks the cytosolic exit of the phosphate and about ~10 Å separate the phosphate from the solvent (Supplementary Fig. 5). We conclude that the structure captures the protein in an 'inward facing occluded state'23.
Inorganic phosphate is located between the two domains buried in the middle of the membrane at a location similar to the substrate binding sites in other Major Facilitators9,13,15 (Fig. 1a). The phosphate is coordinated by Tyr150(M4), Gln177(M5), Trp320(M7), Asp324(M7), Tyr328(M7) and Asn431(M10) as well as by electrostatic interaction from the edge of Phe174(M5) (Fig. 1b). All these residues are fully conserved in the family of Phosphate:H+ symporters. Asp324(M7) coordinates the phosphate with both carboxyl oxygens (Fig. 1b). In Pho84, the corresponding residue (358) is essential for translocation, but initial phosphate-binding is unchanged by its replacement with a asparagine, mimicking a protonated aspartate24. This suggests that the aspartate is protonated before engaging the phosphate. The conserved Lys459(M11) has been proposed to be involved in increasing the affinity for phosphate, with point-mutations causing a 2- to 3-fold decrease in affinity24. In the PiPT structure, Lys459 is located next to the binding site with the lysine side-chain amine ~5 Å from the phosphate, too far away to interact with it (Supplementary Fig. 6). In this configuration, Lys459 could either play a role in initial outward facing phosphate binding, or possibly in charge compensation of Asp324 in the empty form of PiPT.
A tunnel is visible going from the binding site Tyr150 to the cytosol through the N-domain (Fig. 2, Supplementary Fig. 5). This cytosolic tunnel is substantially smaller (smallest diameter 1.2 Å) than phosphate, going from the binding site, between M4 and M1 towards the bottom of M3 and M6 leading to the cytosolic side. In the structure the cytosolic half of M4 is more flexible than the rest of the protein, as reflected in atomic displacement parameters that are almost twice as high as the surrounding residues (185 Å2 vs. 107 Å2) (Fig. 2a, Supplementary Fig. 4e). Related to this flexibility, a conspicuous glycine-motif with four glycines is located at the middle of the M4 helix, introducing mobility by creating a hinge-region (Supplementary Fig. 7a).
Proton transfer through the membrane is expected to involve negatively charged residues12,14,23,25. There are four negatively charged and conserved residues (Asp45(M1), Asp48(M1), Glu108(M3), Asp149(M4)) in the membrane embedded part of PiPT besides the key-residue Asp324 (Fig. 2a). Asp48 interacts with a buried Arg139 (Supplementary Fig. 6) and all the remaining residues are exposed to the cytosolic tunnel (Fig. 2b). In Pho84, the equivalent of Asp149 (178) has been proposed to be involved in transport at a later stage in the transport cycle than Lys459 (492) or Asp324 (358)24. The location of these membrane buried carboxylates implicates the cytosolic tunnel in proton transfer to the cytosol.
The structure suggests a tentative model of phosphate-import by Phosphate:H+ symporters (Fig. 3). In the outward open state, Asp324 and other C-domain residues of the central binding site bind phosphate. Protonation of Asp324 lowers the energy barrier for phosphate binding and this ensures coupling between driving force and substrate translocation23,26. Also, the aspartate helps to select protonated phosphate (phosphate monobasic) versus fully ionized divalent oxyanions like sulphates27–29. Asp324 thus might have a dual role being the proton gatekeeper and ensuring substrate-specificity. As phosphate binds, N-domain residues move in and ensure an optimal fit, thereby repositioning the N-domain to close the entry pathway, forming the outward occluded state. Tyr150 located on the cytosolic side of the M4 glycine-motif interacts with the phosphate in the structure, shifting the flexible region of the M4 helix to form the cytosolic tunnel. Via the tunnel, Asp45, Asp149 and Glu108 create a proton relay from the phosphate binding site to the cytosol that would allow protons to escape, but not permit the passage of phosphate. As positive charge is removed from the binding site along this relay, the binding of phosphate becomes unfavourable and the phosphate exit pathway between the two domains is forced open. In the structure M2 is slightly split apart from M11 at the cytosolic side, and from this conformation, release of the phosphate will require only small rearrangements of M4 and M5 to allow opening of the phosphate exit-pathway lined by the N-domain on one side and the C-domain on the other side (Supplementary Fig. 5). This opening movement seems stalled by 3 salt-bridges formed between the M4-M5 connecting loop and M8 in the structure (Asp159:Arg447, Arg165:Asp381, Arg166:Glu440). The sequential release of protons and then phosphate, with phosphate released between the two domains without major rearrangements is supported by Molecular Dynamics (Supplementary Fig. 8).
The glycine-motif in M4 could help create the suggested proton relay from the binding site to the cytosol and possibly help reposition the N-domain afterwards to allow phosphate to exit. The motif is fully conserved in the PHS family of proton/phosphate transporters (Supplementary Fig. 7a). The multidrug transporter, EmrD11, and the oxalate/formate antiporter, OxlT8, both have glycine-rich motifs in M4 resembling those in PiPT (Supplementary Fig. 7b). Similar conserved motifs are found in sugar MFS transporters such as that found in M4 of the human SLC2 family of glucose facilitative transporters (GLUTs) (Supplementary Fig. 7c). In support of the proposed role of this motif in the inward facing conformation, the M4 helix does not appear as mobile in the outward facing occluded state of the bacterial GLUT homologue XylE15. Further experiments will verify whether the M4-mobility observed here exists in other MFS families.
The PiPT structure is asymmetric in nature, with distinct functionality of the two domains. A similar division is also proposed for LacY, and the Peptide Transporter, PepT(so), where proton translocation is mediated mainly by the C-domain and substrate recognition mainly by the N-domain9,13,25. Conversely, substrate recognition in PiPT is attained almost exclusively by the C-domain, while the mechanistic elements that allow translocation of protons and substrate are found in the more flexible N-domain.
Our proposed phosphate transporter model is compatible with the MFS Rocker-Switch mechanism, but with some notable modifications. It is consistent with an overall symmetry-related movement of the two domains during translocation, but suggests non-symmetrical intra-domain movements in the N-domain to assist proton translocation involving more complex dynamics.
To explore the impact of this structure on human homologues, we constructed homology models of two representative SLC22 members with different charge specificities: The organic cation transporter OCT1 and the organic anion transporter OAT3 (Supplementary Figs. 9 and 10, Supplementary Table 1). Neither model contains a negatively charged residue at the position corresponding to the proton gate-keeper Asp324, in agreement with the observation that SLC22 transporters are not driven by a proton gradient, but more likely by a sodium gradient7. The homology models did not allow us to confidently predict the position of a possible sodium binding-site in OCT1 and OAT3, but the PiPT binding site residues on M7, Tyr328 and Trp320, are highly conserved in the SLC22 family, suggesting that members of this family share a similar substrate binding mechanism utilizing this helix. The charge of Lys459 is conserved at the corresponding position in the organic anion transporters (e.g., Arg454 in OAT3), but is reversed in the organic cation transporters (e.g., Asp474 in OCT1), consistent with a pivotal role in substrate charge specificity30. Finally, the predicted binding pockets in OCT1 and OAT3 are larger than those in PiPT, in agreement with their broader substrate specificities (Supplementary Fig. 9).
In summary, this first structure of a eukaryotic MFS member explains structural/functional relationships of phosphate/proton symport by providing structural evidence for phosphate affinity and specificity and connecting the proton-motive force to phosphate translocation. PiPT provides a strong template for modelling key transporters whose malfunctions in humans are associated with diseases such as cancer and diabetes (e.g., MCT-1 and GLUT4), as well as those that mediate drug absorption, distribution and elimination (e.g., OCT1). These findings provide new insights into charged ligand recognition, binding and release in the context of active membrane transport, a process essential to all living cells.