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 transceptors
1–5
. 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 resistance
6,7
. 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 gradients
1
. Structures of eight bacterial MFS transporters have been determined by 2D and 3D
crystallography
8–15
. Based on the first of these a 'Rocker-Switch' mechanism was proposed
9,10
, 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 site
8,11,13
and a similarly occluded and compact structure for the Lactose Permease (LacY) has
been suggested by molecular dynamics simulations
16
, double electron-electron resonance measurements
17
and homology modeling
18
. 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 growth
19
. 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-plant
20
. PiPT belongs to the Phosphate:H+ Symporter (PHS) family within the Major Facilitator
Super-family
1
. 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 structures
10,13,14
. The linker has no sequence similarity to the four-helix bundle domain observed in
the bacterial GLUT homologue XylE
15
.
The overall conformation of PiPT is similar to structures of MFS transporters solved
in the occluded state
8,11,13
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 Facilitators
9,13,15
(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 aspartate
24
. 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 affinity2
4
. 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 residues
12,14,23,25
. 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 translocation
23,26
. Also, the aspartate helps to select protonated phosphate (phosphate monobasic) versus
fully ionized divalent oxyanions like sulphates
27–29
. 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, EmrD
11
, and the oxalate/formate antiporter, OxlT
8
, 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 XylE
15
. 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-domain
9,13,25
. 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 gradient
7
. 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 specificity
30
. 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.
Methods Summary
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).
Methods
Sample preparation
A 2-micron Saccharomyces cerevisiae expression construct based on p423_GAL1
31
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 described
32
. 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 XDS
33
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 suite
34
.Extensive molecular replacement was attempted essentially as described
35
but was unsuccessful in solving the phase problem. Initial heavy-atom positions of
two platinum sites were found by SIRAS in SHELXC/D
36
using detwinned datasets where the estimated twin-fraction was matched between datasets
and minimized as much as possible
37
. 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 SHARP
38
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 DMMULTI
39
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 data
35,40
. 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 O
41
and refinement in phenix.refine
42
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 reflections
43
. 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 CC1/2
44
of 25%. The final model yielded a crystallographic R-factor of 22.2% and a free R-factor
of 25.9%. MolProbity
45
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 Mole
46
. Electrostatic surfaces were calculated using APBS
47
. All structural figures were prepared using PyMOL
48
.
Homology Modelling
Alignments between sequences and PiPT were calculated using MUSCLE
49
and PROMALS3D
50
, followed by manually refining gaps based on the transmembrane regions observed in
the PiPT structure and predicted for other sequences using Phobius
51
. Homology models of OCT1 (SLC22A1) and OAT3 (SLC22A8) were constructed using MODELLER-9v11
52
and assessed using Z-DOPE
53
, 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 Scwrl4
54
.
Molecular Dynamics
Simulations were performed with GROMACS4
55
, using the CHARMM27
56
all-atom force field and the TIP3P
57
water model. Topology and charges for the phosphate ion were generated with ParamChem.org
58
. PiPT was oriented in an implicit lipid bilayer using PPM
59
, then immersed in a explicit 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC) lipid
bilayer and water using CHARMM-GUI
60
. 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 Berendsen
61
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 Berendsen
61
barostat. Each production run was 100ns long, in the NpT ensemble controlled by the
Bussi-Donadio-Parrinello
62
thermostat and the semi-isotropic Parrinello-Rahman
63
barostat.
Supplementary Material
1
2