Crystal structure of a YeeE/YedE family protein engaged in thiosulfate uptake

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Science Advances  26 Aug 2020:
Vol. 6, no. 35, eaba7637
DOI: 10.1126/sciadv.aba7637
  • Fig. 1 Thiosulfate uptake depending on YeeE in E. coli.

    (A) Uptake of sulfate and thiosulfate in the inner membrane. (B to E) Survivability of E. coli WT (MG1655) and its derivatives (ΔcysPUWA and ΔcysPUWA ΔyeeE) in nonsulfur medium (B) or single sulfur source minimal media containing 100 μM sulfate (C), 500 μM thiosulfate (D), or 100 μM cysteine (E). The OD600 (optical density at 600 nm) was monitored every hour. (F) Growth complementation of ΔcysPUWA ΔyeeE (DE3) in the single sulfur source minimal media containing thiosulfate by expression of E. coli YeeE. OD600 was monitored every 30 min. Error bars indicate the SD (n = 3). The growth patterns shown in (B) to (F) indicate that YeeE transports thiosulfate.

  • Fig. 2 Crystal structure of YeeE.

    (A and B) Overall structure of StYeeE in cartoon (A) and ribbon (B) representations from the membrane side. The transparency of H1, H3, H8, and H10 in (A) is 30% to clearly display the inside. Numbers of α helices are indicated, and YeeE characteristic loops (LA–LD) are highlighted in red and magenta. Thiosulfate ions are shown as a space-filling model. (C) YeeE structure in ribbon representation from the outside. (D) Close-up view of thiosulfate-binding site. 2FoFc map and thiosulfate-omit (FoFc) map are shown with 1.2 σ and 4.0 σ, respectively. (E) Surface model of YeeE viewed from the outside, colored to indicate electrostatic potential ranging from blue (+10 kT/e) to red (−10 kT/e). (F) Schematic topology model of YeeE. YeeE has four short α helices (H2, H5, H9, and H12), the LA–LD loop, and nine transmembrane α helices (H1, H3, H4, H6, H7, H8, H10, H11, and H13).

  • Fig. 3 Intramolecular pseudo 222 fold in YeeE.

    (A) Structure of YeeE with three orthogonal pseudo dyad axes a, b, and c. The pseudo symmetrical elements H1–H3, H4–H6, H8–H10, and H11–H13 are colored in orange, green, yellow, and blue, respectively. (B) Each intramolecular pseudo twofold symmetry. YeeE structure viewed along one of the pseudo twofold axes (top, a axis; middle, b axis; bottom, c axis). Each pair of pseudo twofold symmetry elements are shown, e.g., H1–H3 and H4–H6 (top). (C) Superimposition of the H1–H3, H4–H6, H8–H10, and H11–H13. (D) Structure-based sequence alignment of H1–H3, H4–H6, H8–H10, and H11–H13, generated from (C).

  • Fig. 4 Conserved residues and crystallographic B factor of YeeE.

    (A) Mapping of conserved residues onto the crystal structure of YeeE. The amino acids are colored according to their ConSurf conservation grades, as shown in fig. S1. (B) Crystallographic B factor of YeeE. The crystal structure of YeeE is colored ranging from 20 to 80 Å2. The top and bottom figures show the full-length structures and H1- and H8-omitted structures, respectively.

  • Fig. 5 Mutational analysis and working model of YeeE.

    (A) Complementation of ΔcysPUWA ΔyeeE (DE3) growth by the indicated YeeE mutants as in Fig. 1F. Growth (ΔOD600) at 24 hours was normalized to that of WT YeeE-expressing cells. Error bars indicate the SD (n = 3). (B) Mapping of the mutational analysis. The side chains of the mutated positions in (A) are shown as a stick model. The orange and blue residues are essential and nonessential, respectively. (C) Cross-sectional model of YeeE along the dashed line in Fig. 2E. The LA–LD loops are shown as a tube model. Thiosulfate, conserved R215, and three cysteines in the loops are shown as a stick model. The thiosulfate-omit (FoFc) map of WT and C91A mutant with 4.0 σ are shown in blue and green mesh, respectively. Position I is the thiosulfate-binding site. Positions II and III are predicted binding sites. (D) Schematic working model of YeeE. The LA–LD loops and their vicinity helices are shown. The conserved arginine in the LC and three conserved cysteines in the LA, LB, and LD are depicted. Thiosulfate ions may be recognized at position I and passed through the membrane along the arrow.

  • Table 1 Data collection and refinement.

    Statistics for the highest-resolution shell are shown in parentheses.

    PDB IDYeeE (WT)YeeE (C91A)SeMet YeeE
    Data collection
    X-ray sourceSPring-8
    Wavelength (Å)1.441.000.979
    Space groupC2221C2221C2221
    a, b, and c (Å)73.51, 95.32,
    and 101.4
    73.91, 94.61,
    and 101.5
    73.60, 95.68,
    and 101.5
    Resolution range
    No. of merged
    515 × (10°,
    Δφ = 0.100)
    93 × (10°,
    Δφ = 0.100)
    326 × (7°,
    Δφ = 0.100)
    Total reflections2,037,612
    12,366 (1224)10,299 (898)9,125 (901)
    Multiplicity164.8 (163.8)29.3 (17.1)73.8 (76.5)
    99.85 (99.59)89.62 (76.73)99.84 (99.67)
    I/σ(I)20.49 (2.31)10.15 (1.28)19.40 (5.31)
    R-meas0.6336 (8.719)0.3629 (3.059)0.3533 (1.72)
    CC1/20.994 (0.522)0.981 (0.213)0.997 (0.859)
    No. of reflections12,353 (1219)10,104 (841)
    No. of atoms2,7842,523
    RMS derivations
    Bond length (Å)0.0020.002
    Bond angles (°)0.490.56
    Average B factor36.5543.75

Supplementary Materials

  • Supplementary Materials

    Crystal structure of a YeeE/YedE family protein engaged in thiosulfate uptake

    Yoshiki Tanaka, Kunihito Yoshikaie, Azusa Takeuchi, Muneyoshi Ichikawa, Tomoyuki Mori, Sayaka Uchino, Yasunori Sugano, Toshio Hakoshima, Hiroshi Takagi, Gen Nonaka, Tomoya Tsukazaki

    Download Supplement

    The PDF file includes:

    • Figs. S1 to S5

    Other Supplementary Material for this manuscript includes the following:

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