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

Abstract

We have demonstrated that a bacterial membrane protein, YeeE, mediates thiosulfate uptake. Thiosulfate is used for cysteine synthesis in bacteria as an inorganic sulfur source in the global biological sulfur cycle. The crystal structure of YeeE at 2.5-Å resolution reveals an unprecedented hourglass-like architecture with thiosulfate in the positively charged outer concave side. YeeE is composed of loops and 13 helices including 9 transmembrane α helices, most of which show an intramolecular pseudo 222 symmetry. Four characteristic loops are buried toward the center of YeeE and form its central region surrounded by the nine helices. Additional electron density maps and successive molecular dynamics simulations imply that thiosulfate can remain temporally at several positions in the proposed pathway. We propose a plausible mechanism of thiosulfate uptake via three important conserved cysteine residues of the loops along the pathway.

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. 2F_o − F_c map and thiosulfate-omit (F_o − F_c) 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 Ų. 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 (F_o − F_c) 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.