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. 2013 Apr 25;496(7446):533-6.
doi: 10.1038/nature12042. Epub 2013 Mar 31.

Crystal Structure of a Eukaryotic Phosphate Transporter

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Free PMC article

Crystal Structure of a Eukaryotic Phosphate Transporter

Bjørn P Pedersen et al. Nature. .
Free PMC article

Abstract

Phosphate is crucial for structural and metabolic needs, including nucleotide and lipid synthesis, signalling and chemical energy storage. Proton-coupled transporters of the major facilitator superfamily (MFS) are essential for phosphate uptake in plants and fungi, and also have a function in sensing external phosphate levels as transceptors. Here we report the 2.9 Å structure of a fungal (Piriformospora indica) high-affinity phosphate transporter, 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. 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.

Figures

Figure 1
Figure 1. Structure of the High Affinity Phosphate Transporter, PiPT
The structure represents an inward facing occluded state of the phosphate transporter in complex with phosphate. a, Phosphate (shown as spheres) is buried in the membrane at the interface between the N-domain (pale green) and C-domain (blue). Selected residues are shown as sticks. Black bars depicts the approximate location of the membrane. b, The phosphate binding site with yellow dashes indicate possible hydrogen bonds (2.2–3.8 Å distances) to phosphate. Top, The omit mFobs-DFcalc density for phosphate is contoured in orange (4σ). Bottom, The 2mFobs-DFcalc density for phosphate and selected M7 residues is contoured in red (2σ). Other residues are omitted for clarity (Supplementary Fig. 4e).
Figure 2
Figure 2. The proposed proton exit pathway
a, All carboxylates are shown as yellow sticks. Four carboxylate side chains in addition to Asp 324 lie in the transmembrane region and all surround the observed tunnel. The tunnel, putatively an proton conductance pathway, is too narrow for phosphate (smallest diameter 1.2 Å). The M4 helix which forms part of the tunnel has high flexibility, indicated by the atomic displacement coded by thickness of the main-chain and a colour-gradient from blue (low disorder) to red (high disorder). The average atomic displacement parameter is 107 Å2 for the protein chain and 185 Å2 for the cytosolic half of the M4 helix (Supplementary Fig. 4f). b, Rotated 90° about the vertical axis the carboxyls line the cytosolic tunnel. c, Electrostatic (−5 to 5 kT/e) surface representation of PiPT (cytosolic side) highlights the negative potential found in the tunnel. The phosphate oxygens are visible via the tunnel.
Figure 3
Figure 3. Proposed mechanism of phosphate transport
Asp324 is the central proton donor/acceptor of the transporter, found at the centrally located binding site (red) in the inward occluded state described (center). Models of outward open (left) and inward open (right) forms of PiPT were made by structural alignment with the fucose/proton symporter (pdb 3O7Q) and the Lactose permease (pdb 2CFQ) respectively. In the outward open conformation, the protonated form of Asp324 (1) gives preference to phosphate binding (2). Optimal binding of phosphate requires Gln177 and possible other residues to pull the N-domain towards the binding site (3), forming an outward occluded state. Conformational movements (4) opens up the tunnel in the inward occluded state (5). Exposure of the negatively charged cytosolic tunnel pulls a proton from the binding site (6) and the resulting repulsion between phosphate and the now deprotonated binding site (7) allows phosphate to exit to the cytosol between the two domains (8).

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