Research ArticleBIOCHEMISTRY

Crystal structures of a ZIP zinc transporter reveal a binuclear metal center in the transport pathway

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Science Advances  25 Aug 2017:
Vol. 3, no. 8, e1700344
DOI: 10.1126/sciadv.1700344
  • Fig. 1 Overall structure of BbZIP.

    (A) Side views of BbZIP in the putative membrane with labeled TMs (α1 to α8). The protein is shown in cartoon mode and colored in rainbow with the N terminus in blue and the C terminus in red. The bound Cd2+ ions are shown as yellow-brown spheres. The dotted lines indicate the disordered interhelical loops (the loop between TM3 and TM4 and the loop between TM7 and TM8). (B) Top view of BbZIP in the direction indicated by the black arrow in (A). The two Cd2+-binding sites in the transport pathway are labeled as M1 and M2, respectively. (C) Cross-sectional view of the electrostatic potential map of BbZIP. The two highly negatively charged cavities are indicated by arrows. Note that the pathway from the entrance cavity to the metal-binding sites is blocked. (D) Electrostatic potential map of the exit cavity.

  • Fig. 2 Metal-binding sites in BbZIP.

    (A) Cd2+ binding to BbZIP. The protein is in cartoon mode and colored in rainbow. The four Cd2+-binding sites are labeled (M1 to M4). The bound Cd2+ ions are shown as yellow-brown spheres. The region in the dotted frame is shown in (B). (B) Zoomed-in view of the binuclear metal center in the direction indicated by the arrow in (A). The blue meshes show the anomalous difference map of Cd2+ ions at M1 and M2 (σ = 5). The data set was collected at 1.7969 Å, where Cd generates much stronger anomalous signals than at 0.9792 Å. The metal-chelating residues are labeled and shown in stick mode with the same colors as in (A). The dashed lines indicate the bonding of Cd2+ ions with the coordinating residues in the range of 2.6 to 2.9 Å. The ordered water molecules (W) are shown as small red spheres. (C) Metal binding to Zn2+-substituted BbZIP. The seven metal-binding sites are labeled (M1 to M7). The bound Zn2+ and Cd2+ ions are shown as gray and yellow-brown spheres, respectively. The region in the dotted frame is shown in (D). (D) Zoomed-in view of the binuclear metal center in the direction indicated by the arrow in (C). The green meshes show the FoFc omit map (σ = 5) of the metals at M1 and M2. The blue meshes (σ = 5) and pink meshes (σ = 3) show the anomalous different maps calculated from the data sets collected at 1.2782 and 1.3190 Å, respectively. The K-edge of zinc is 1.2835 Å. The metal-chelating residues are labeled and shown in stick mode with the same colors as in (C). The dashed lines indicate the bonding of Zn2+ and Cd2+ ions with the coordinating residues within the range of 2.0 to 2.2 Å (for Zn2+) and 2.6 to 2.8 Å (for Cd2+). The ordered water molecules are shown as small red spheres.

  • Fig. 3 Structural model and functional characterization of hZIP4.

    (A) Model structure of the TMD of hZIP4. Homology modeling was conducted using the SWISS-MODEL server. The protein is shown in cartoon mode. The residues mutated in AE are labeled and highlighted in red. The region in the dotted frame is shown in (C). (B) Sequence alignment of BbZIP and all 14 human ZIPs within the portions of TM4 and TM5. The potential metal-chelating residues contributive to M1 or M2 are bolded and highlighted in red. The bridging residue is involved in both metal-binding sites. The highly conserved residues are drawn in white on black background, and the generally conserved residues are drawn on gray background. (C) Zoomed-in view of the modeled binuclear metal center in hZIP4. The metal-chelating residues are colored as in Fig. 2 for comparison. The modeled Zn2+ ions are depicted as gray spheres. The dashed lines indicate the bonding of Zn2+ with the coordinating residues within the range of 2.3 to 3.1 Å. (D and E) Alanine scanning (D) and arginine scanning (E) of the metal-chelating residues at the putative binuclear metal center of hZIP4. The H379A/D375A double mutant is also included in (E). The activities of the mutants are expressed as relative activity compared with the wild-type (WT) protein. Each data point represents a mean of a total of nine calibrated relative activities from three independent experiments. The error bars indicate 1 ± SD; *P < 0.01. The data of three independent experiments, including zinc uptake, total expression, and surface expression of each mutant in human embryonic kidney (HEK) 293T cells, are shown in fig. S15. Note that the relative activity of the H540R variant was underestimated in this assay by approximately 30% because of its significantly reduced affinity toward zinc (fig. S12B).

  • Fig. 4 Proposed metal transport pathway of BbZIP.

    A side view of the electrostatic potential surface of BbZIP is shown in a putative membrane (solid lines). The solid arrows indicate the proposed metal transport pathway connected by a series of metal-binding sites, whereas the dotted arrow indicates a putative pathway at the extracellular side, which is currently blocked by hydrophobic residues from TM2 (M99 and A102), TM5 (L200 and I204), and TM7 (M269; not shown for clarity). The metal substrate is depicted as light blue spheres. The involved residues in stick mode are labeled. Note that H177 may adopt two conformations, implying a role in metal release from M1.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/8/e1700344/DC1

    fig. S1. Crystal structure of BbZIP.

    fig. S2. Crystal packing of zinc-substituted BbZIP in two perpendicular directions.

    fig. S3. Size exclusion chromatography of BbZIP in DDM.

    fig. S4. Multiple sequence alignment of bacterial ZIPs.

    fig. S5. A novel fold of BbZIP.

    fig. S6. Tilted structure of BbZIP in the putative membrane.

    fig. S7. Metal content analysis of the purified BbZIP by ICP-MS.

    fig. S8. Structural comparison of M99.

    fig. S9. Anomalous difference maps at M3 and M4 in the zinc-substituted structure.

    fig. S10. Sequence alignment of BbZIP with hZIP4 TMD.

    fig. S11. Comparison of hZIP4 structure models.

    fig. S12. Determination of the zinc uptake kinetic parameters.

    fig. S13. Proposed conformational change facilitated by metal release from the binuclear metal center.

    fig. S14. The distances between the two metal-binding sites (M1 and M2) and S106.

    fig. S15. Three independent zinc uptake assays.

    fig. S16. Time-dependent zinc uptakes by HEK293T cells overexpressing hZIP4.

    table S1. Crystallographic statistics.

    table S2. Strains, plasmids, and primers.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Crystal structure of BbZIP.
    • fig. S2. Crystal packing of zinc-substituted BbZIP in two perpendicular directions.
    • fig. S3. Size exclusion chromatography of BbZIP in DDM.
    • fig. S4. Multiple sequence alignment of bacterial ZIPs.
    • fig. S5. A novel fold of BbZIP.
    • fig. S6. Tilted structure of BbZIP in the putative membrane.
    • fig. S7. Metal content analysis of the purified BbZIP by ICP-MS.
    • fig. S8. Structural comparison of M99.
    • fig. S9. Anomalous difference maps at M3 and M4 in the zinc-substituted structure.
    • fig. S10. Sequence alignment of BbZIP with hZIP4 TMD.
    • fig. S11. Comparison of hZIP4 structure models.
    • fig. S12. Determination of the zinc uptake kinetic parameters.
    • fig. S13. Proposed conformational change facilitated by metal release from the binuclear metal center.
    • fig. S14. The distances between the two metal-binding sites (M1 and M2) and S106.
    • fig. S15. Three independent zinc uptake assays.
    • fig. S16. Time-dependent zinc uptakes by HEK293T cells overexpressing hZIP4.
    • table S1. Crystallographic statistics.
    • table S2. Strains, plasmids, and primers.

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