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Nature. Author manuscript; available in PMC Oct 25, 2013.
Published in final edited form as:
PMCID: PMC3678552
NIHMSID: NIHMS449606
Crystal structure of a eukaryotic phosphate transporter
Bjørn P. Pedersen,1 Hemant Kumar,1,2 Andrew B. Waight,1 Aaron J. Risenmay,1 Zygy Roe-Zurz,1 Bryant H. Chau,1 Avner Schlessinger,3 Massimiliano Bonomi,3 William Harries,1 Andrej Sali,3 Atul K. Johri,2 and Robert M. Stroud1
1Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94158, United States of America.
2School of Life Sciences, Jawaharlal Nehru University, New Delhi-110067, India.
3Department of Bioengineering and Therapeutic Sciences, Department of Pharmaceutical Chemistry, California Institute for Quantitative Biosciences, University of California, San Francisco, CA 94158, United States of America.
Correspondence and requests for materials should be addressed to R.M.S. (stroud/at/msg.ucsf.edu
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 transceptors15. 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 crystallography815. 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.
Figure 1
Figure 1
Structure of the High Affinity Phosphate Transporter, PiPT
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).
Figure 2
Figure 2
The proposed proton exit pathway
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 sulphates2729. 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).
Figure 3
Figure 3
Proposed mechanism of phosphate transport
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.
The Piriformospora indica high affinity phosphate transporter PiPT (accession number A8N031) was expressed in Saccharomyces cerevisiae and purified using a polyhistidine affinity-tag. Solubilization and purification used dodecyl-beta-D-maltoside followed by n-nonyl-beta-D-glucoside. Crystals were grown by vapour diffusion. Crystallographic data were collected at the Advanced Light Source beamline 8.3.1 and later at Advanced Photon Source beamline 23-ID-B and 23-ID-D. All crystals showed clear signs of hemihedral twinning with a variable twin-fraction from 0.14 to 0.47. Experimental phases were determined using derivative crystals containing K2PtCl6 or (Ta6Br12)Br2. Heavy-atom derived phases were refined and extended to the maximum resolution of the native data by density modification, exploiting histogram mapping, solvent flattening with a solvent content of 73%, two-fold non-crystallographic symmetry and four-fold inter-crystal averaging. Final refinement using data to 2.9 Å resolution produced a structural model with a crystallographic R-factor of 22.2% and a free R-factor of 25.9% (Supplementary Table 2).
Sample preparation
A 2-micron Saccharomyces cerevisiae expression construct based on p423_GAL131 contained nucleotides coding for the Piriformospora indica high affinity phosphate transporter PiPT (accession number A8N031) as well as N-terminal and C-terminal purification-tags as described32. Transformed S. cereviciae (strain DSY-5) were grown in a 15 l culture vessel (Biostat C15L Sartorius AG) to high density and induction was done in fed-batch using 40% galactose, and harvested after a 16 hour induction. Harvested yeast were washed in cold water, spun down and resuspended in Lysis-buffer (100 mM Tris pH 7.5, 600 mM NaCl, 1 mM EDTA, 1 mM tris(2-carboxyethyl)phosphine (TCEP), 1.2 mM phenylmethylsulfonyl fluoride) before lysis by bead beating using 0.5 mm glass-beads. The homogenate was centrifuged for 25 minutes at 21,600 g, followed by sedimentation of membranes by ultracentrifugation at 185,000 g for 150 minutes. Membrane pellets were resuspended in Membrane-buffer (50 mM Tris pH 7.5, 500 mM NaCl, 20% Glycerol) before being frozen in liquid nitrogen in 3 g aliquots. A normal yield was 20–25 g membrane-pellet from 100 g cells. 3 g membrane were solubilized for 30 minutes in Membrane-buffer using 300 mg n-dodecyl-beta-D-maltoside (DDM) (1:10 (w/w) ratio) in a total volume of 50 ml, after which unsolubilized material was removed by filtration using a 1.2 µm filter. 20 mM imidazole pH 7.5 were added and the solubilized membranes loaded on a pre-equilibrated 1 ml Ni-NTA column (GE Healthcare) at 1 ml/min. After loading, the column was washed with 20 column-volumes of W100-buffer (Membrane buffer supplemented with 100 mM Imidazole pH 7.5, 1 mM TCEP, 10 mM K2HPO4 and 0.05% DDM), and eluded in 5 ml G-buffer (50 mM MES pH 6.5, 200 mM NaCl, 10 mM K2HPO4, 0.5 mM TCEP, 0.2% n-nonyl-beta-D-glucoside (NG)) supplemented with 500 mM Imidazole pH 7.5. To remove purification tags, Bovine Thrombin and HRV 3C Protease were added and the sample dialyzed for 16 hours against 100 ml G-buffer. A normal yield was 10 mg pure PiPT from 3 g of membrane. After dialysis the sample was concentrated using a spin-column (50 kDA cut-off, Amicon) to 500 µl and injected on a size-exclusion column (Superdex 200, GE Healthcare) pre-equilibrated in G-buffer. Peak fractions were concentrated to 10–15 mg/ml before a new dialysis for 16 hours against 100 ml G-buffer. An ultracentrifugation spin (108,000 g, 20 minutes) was applied before crystallization setup.
Crystal Growth
Crystals were grown at 20°C by vapour diffusion in 2+2 µl hanging drops with a reservoir containing 26–29% (w/v) Pentaerythritol propoxylate (5/4 PO/OH), 6–11% Polyethylene glycol 400, 200 mM KCl and 100 mM Sodium Citrate pH 5.5. Hexagonal crystals, with a final size of around 200x200x50 µm, were obtained after typically one week of crystal-growth but would be extremely sporadic in appearing (Supplementary Fig. 2a). Crystals grew in DDM, DM and NG, but morphology and space group changed in NG, and diffraction improved from 4–6 Å to 2.9 Å in the best case. Data were collected at the Advanced Light Source beamline 8.3.1 and later at the Advanced Photon Source beamline 23-ID-B and 23-ID-D. Initial crystals displayed ~10 Å resolution, with severely split spots. Several lines of crystal improvement augmented diffraction properties. Optimized crystals normally diffracted isotropically to 3.2 Å, with a single crystal screened extending to 2.9 Å (Supplementary Fig. 2a). Heavy-atom derivatives were obtained by adding K2PtCl6 or (Ta6Br12)Br2 to the crystals a day prior to flash-cooling, either as salt or as a concentrated, aqueous solution.
Data processing
Data sets were processed using XDS33 in space group R 3. The data showed clear signs of hemihedral twinning with the twin law (k,h,-l) (Supplementary Table 2). The estimated twin fraction varied from 0.14 to 0.47. The data was detwinned using DETWIN from the CCP4 suite34.Extensive molecular replacement was attempted essentially as described35 but was unsuccessful in solving the phase problem. Initial heavy-atom positions of two platinum sites were found by SIRAS in SHELXC/D36 using detwinned datasets where the estimated twin-fraction was matched between datasets and minimized as much as possible37. A single Ta6Br12-cluster site were identified using the initial platinum phases, and experimental MIRAS phases combining two platinum sites and one Ta6Br12-cluster site were calculated to 3.5 Å in SHARP38 using detwinned datasets (Supplementary Fig. 2b). Gross map-features (e.g. number of discernible α-helices) were significantly improved when experimental phases and maps were calculated from detwinned data as opposed to twinned data. Heavy-atom-derived phases were refined, combined and extended at the maximum resolution of the native data by density modification in DMMULTI39 exploiting histogram mapping, solvent flattening with a solvent content of 73%, two-fold non-crystallographic symmetry and four-fold inter-crystal averaging. It was helpful to start phase extension at low resolution (10 Å) and very gradually extend (2000 cycles) to the full resolution of the data35,40. The resulting electron-density map was of good quality given the low phasing power, providing a continuous trace of the main-chain (Supplementary Table 2, Supplementary Fig. 4). Iterative model building in O41 and refinement in phenix.refine42 gradually improved the model and the fit to the experimental map. At later stages model-building was guided by 2mFo-dFc maps using model-phases. Final refinement in phenix.refine used 2-fold torsion-NCS with a refinement strategy of individual sites, individual ADP, occupancy (phosphate only) and TLS (4 groups), against a maximum likelihood (ML) target with reflections in the 69–2.9 Å range of the detwinned dataset (Supplementary Table 2). Refinement could also be done directly against the twinned data using a modified least-squares target ('twin_lsq_f'), but the resulting model was of poorer quality as reflected in worse R-factors and poorer electron-density in the maps for omitted regions. This might be caused by the usage of a least-squares target which disfavours the usage of weak reflections43. The final resolution cut-off was based on the behaviour of the crystallographic R-factor (Rwork) in 0.1 Å resolution shells to ensure full utilization of the data. We deemed that an Rwork below 40% meant the data in the given resolution shell was effectively contributing beneficially to the model. The selected cut-off of 2.9 Å based on this approach correlated with an intra-dataset correlation coefficient CC1/244 of 25%. The final model yielded a crystallographic R-factor of 22.2% and a free R-factor of 25.9%. MolProbity45 evaluation of the Ramachandran plot gave 89.5% in favoured regions, and 2.6% outliers as expected for this resolution. The cytosolic tunnel was visualized using Mole46. Electrostatic surfaces were calculated using APBS47. All structural figures were prepared using PyMOL48.
Homology Modelling
Alignments between sequences and PiPT were calculated using MUSCLE49 and PROMALS3D50, followed by manually refining gaps based on the transmembrane regions observed in the PiPT structure and predicted for other sequences using Phobius51. Homology models of OCT1 (SLC22A1) and OAT3 (SLC22A8) were constructed using MODELLER-9v1152 and assessed using Z-DOPE53, a normalized atomic distance-dependent statistical potential based on known protein structures. Side-chains of selected residues in the initial OCT1 (Lys214 and Asp474) and OAT3 (Asn450 and Arg454) models were then refined using Scwrl454.
Molecular Dynamics
Simulations were performed with GROMACS455, using the CHARMM2756 all-atom force field and the TIP3P57 water model. Topology and charges for the phosphate ion were generated with ParamChem.org58. PiPT was oriented in an implicit lipid bilayer using PPM59, then immersed in a explicit 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) lipid bilayer and water using CHARMM-GUI60. Periodic boundary conditions and a triclinic box with volume of 557.7 nm3 were used. PipT was simulated a) at neutral pH and b) with two protons added to Asp45 and Asp324. Equilibration was performed by three 10ns long runs, gradually increasing the temperature from 100K to 300K, in the canonical (NVT) ensemble controlled by the Berendsen61 thermostat. The positions of non-hydrogen atoms of PiPT were restrained by a harmonic potential, with gradually decreasing intensity. A final equilibration step was carried out for 10ns without restraints, in the isothermal–isobaric (NpT) ensemble controlled by the semi-isotropic Berendsen61 barostat. Each production run was 100ns long, in the NpT ensemble controlled by the Bussi-Donadio-Parrinello62 thermostat and the semi-isotropic Parrinello-Rahman63 barostat.
Supplementary Material
Acknowledgements
We thank Kathleen Giacomini for discussions about SLC transporters; Poul Nissen for helpful comments that improved the manuscript; James Holton, George Meigs, Craig Ogata and Nagarajan Venugopalan for assistance with synchrotron data collection at the Advanced Light Source and Advanced Photon Source; and Christopher Waddling, Pascal Wassam and Meseret Tessema for technical assistance. B.P.P. was supported by a Postdoctoral fellowship from the Carlsberg Foundation and later by a fellowship from the Danish Cancer Society; H.K. by a fellowship from Woods Whelan foundation, Council of Scientific and Industrial Research, Govt. of India and a travel grant from Jawaharlal Nehru University, New Delhi.; A.Sc by NIH postdoctoral fellowship F32 GM088991; A.S. by NIH grants U54 GM094625 and U01 GM61390; A.K.J. by a Research Assistant Professorship to do work at UCSF from the American Society for Microbiology; and R.M.S. by NIH grants U54 GM094625, GM24485 and GM073210.
Footnotes
Author Contributions
B.P.P. did expression, purification and crystallization experiments, collected and processed the data, and determined, refined and analysed the structure. H.K. identified the target, did purification and crystallization experiments, collected data and identified the use of NG for crystallization. A.B.W. optimized the yeast expression system, and assisted in data collection and data analysis. A.R. helped with protein purification and crystallization. Z.R-Z. assisted in optimization of the yeast expression system, cloned, expressed, purified and characterized the target, and set up initial crystallization experiments. B.C. did cloning, expression tests and cell growth. A.Sc. performed bioinformatics analysis and built human homology models. M.B. did molecular dynamics and analysed the results. W.H. trained H.K and assisted H.K. in data collection. A.S. supervised homology modelling, bioinformatics analysis and Molecular Dynamics. A.K.J. identified the target and initiated the project. R.M.S. supervised the project and analysed the structure. B.P.P. and R.M.S. wrote the paper with input from H.K., A.B.W., A.Sc., A.S. and A.K.J.
Author Information
Coordinates and structure factors have been deposited in the Protein Data Bank with the accession number 4J05. Reprints and permissions information is available at www.nature.com/reprints.
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