Research ArticleSTRUCTURAL BIOLOGY

Structure of the sodium-dependent phosphate transporter reveals insights into human solute carrier SLC20

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Science Advances  07 Aug 2020:
Vol. 6, no. 32, eabb4024
DOI: 10.1126/sciadv.abb4024
  • Fig. 1 Structure of the TmPiT-Na/Pi complex.

    (A) Topology of TmPiT with 12 transmembrane helices that are divided into a transport domain with two inverted topology repeats, N-PD001131 (TM1 to TM3 and HP1a-HP1b, in magenta) and C-PD001131 (TM6 to TM8 and HP2a-HP2b, in blue), and a scaffold domain (TM4/5, in yellow). (B) Ribbon diagram of the TmPiT-Na/Pi complex consisting of a transport domain with N-PD001131 (in magenta) and C-PD001131 (in blue) and a scaffold domain (in yellow). The Pi and Na ions are shown in CPK mode and as purple spheres, respectively. The transmembrane helices in (B) are colored and numbered based on (A). (C) Ribbon diagram of the TmPiT dimer (in bright blue and cyan for subunits A and B, respectively) with height and width dimensions of 61 and 80 Å, respectively. (D) Sequence alignment between N-PD001131 and C-PD001131 for the four highly conserved motifs (boxed and labeled 1 to 4, respectively): GΦNDΦ, GxxxxGxxVxxT, PΦSxT, and IxxxWΦ (x, any amino acid; Φ, hydrophobic residue) are boxed. (E) The electrostatic surface potential is shown for the TmPiT-Na/Pi complex (red, blue, and white for negative, positive, and neutral potentials, respectively). To show the binding pocket, Pi is shown as a sphere, and loops L7 and LHP2 are displayed as ribbons.

  • Fig. 2 Na/Pi binding.

    (A) Ligand selectivity of TmPiT was determined by a microscale thermophoresis binding assay. n.d., not determined. (B) Phosphate (32P) uptake activity of TmPiT was measured using proteoliposomes containing TmPiT. The data in (A) and (B) represent the means ± SD of three independent experiments. (C) Pi- and Na-binding pocket. The Fobs-Fcalc (observed and calculated structure factors) electron density maps of Pi and Na are shown at 8σ and 6σ, respectively. The transmembrane helices TM1, TM6, HP1a-HP1b, and HP2a-HP2b (labeled) are involved in Pi and Na binding. The Pi- and Na-binding residues are shown in CPK mode and as purple spheres, respectively. (D) Zoomed-in view of the Pi-2Na–binding pocket, showing interacting residues. (E) Zoomed-in view of Nafore binding, showing the pentacoordination residues.

  • Fig. 3 Mutational study of the residues involved in Na coordination and conformational change.

    (A) Residues D22 (TM1), D258 (TM6), and D327(HP2) are the three residues interacting with Na1, Na2, and Nafore, respectively. (B) D22A and D258A mutant proteins were reconstituted into proteoliposomes to determine their phosphate (32P) uptake activity. (C) Phosphate bindings of the TmPiT mutants D22A, D258A, D22/258A, and D327Q were determined by microscale thermophoresis. The conformational change of subunits A and B around the inner gates is shown in (D) and (E), respectively. Two inverted repeated domains are shown: HP1 from N-PD001131 (in magenta in both subunits) and HP2 and TM8 from C-PD001131 (in bright blue and cyan for subunits A and B, respectively). The structures shown include Pi and Na, and residues K314 and W378 are labeled. The Pi and Na ions are shown in CPK mode and as purple spheres, respectively. Residues K314 and W378 are shown in CPK mode. The accessible volumes of the exit region were calculated using CASTp (Computed Atlas of Surface Topography of proteins) (27) and are shown in brown. (F) Phosphate bindings of the TmPiT mutants W139A and W378A were determined by microscale thermophoresis. The data in (B), (C), and (F) represent the means ± SD of three independent experiments.

  • Fig. 4 A working model for Na/Pi transport in TmPiT.

    The proposed elevator-like mechanism includes four sequential states: outward open, outward occluded, inward occluded, and inward open. The TmPiT-Na/Pi complex (this study) exists as the inward occluded state. The structure shows the transport domains of the two inverted repeats (N-PD001131, magenta; C-PD001131, blue), the scaffold domain (gray), and Pi and Na, as well as TM3/8 and the L2/LHP1 and L7/LHP2 loops. “W” represents Trp139 and Trp378 in TM3 and TM8, respectively.

  • Fig. 5 Topology and homology modeling of hPiT2 and disease-related variants.

    (A) The topology model of hPiT2 was created on the basis of TmPiT. hPiT2 variants linked to brain calcification disease are colored and grouped into six categories: Pi-binding (orange), Na-binding (green), N-PD001131 (pink), C-PD001131 (blue), dimer (yellow), and S domain (brown). (B) The variants were mapped onto the modeled hPiT2 structure and are shown as spheres, colored according to the system in (A). Residue numbers for human/Tm are also labeled (see details in table S4). The Pi- and Na-binding sites are indicated with dashed outlines.

  • Table 1 Structural and functional comparisons of the TmPiT and hPiT mutants.

    *Values present percentage of wild type. †Pi uptake by hPiT mutants is abolished. ‡These variants are found in human patients with primary familial brain calcification (PFBC) disease.

    TmPiT mutationsFunctional sitePi binding (this study)Pi uptake* (this study)hPiT mutations†hPiT PFBC variants‡ (10, 28)
    D22ANa1/Pi binding22.5 μM4.7%D28N (20)D28N
    D258ANa2/Pi binding27.8 μM18.9%D506N (20)
    D22A/D258ANa1/Na2/Pi bindingn.d.
    D327QNafore bindingn.d.E575Q (26)E575K
    W378AConformational changen.d.W626 duplication
    W139AMay be associated with conformational change7.2 mM

Supplementary Materials

  • Supplementary Materials

    Structure of the sodium-dependent phosphate transporter reveals insights into human solute carrier SLC20

    Jia-Yin Tsai, Chen-Hsi Chu, Min-Guan Lin, Ying-Hsuan Chou, Ruei-Yi Hong, Cheng-Yi Yen, Chwan-Deng Hsiao, Yuh-Ju Sun

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    This PDF file includes:

    • Figs. S1 to S6
    • Tables S1 to S4

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