The outward-facing conformation contains D-galactonate occluded within the substrate recognition site.

(A) Close-up stereo view of the substrate recognition site. Residues Q164, Q264, S370, and N393 form hydrogen bonds (orange dashes) with the 5 hydroxyl groups in D-galactonate. Y44 and Y79 form hydrogen bonds, and Arg47 forms a salt bridge with the carboxyl group of D-galactonate. The Fo-Fc density map of D-galactonate (green mesh) was contoured at 3σ, and the 2Fo-Fc density map (gray mesh) of DgoT residues was contoured at 1σ. (B) The overall structure of DgoT in cylinder representation defines the views shown in panel A (black rectangle), panel C (purple), and panel D (orange). N- and C-terminal domains are shown in blue and green, respectively. ICH1 is shown in cyan and ICH2 is shown in bright green. D-galactonate (yellow) is shown in stick representation. (C) Hydrophobic gating residues F137 and W373 interact with the substrate while forming contacts between N- and C-terminal domains. N141 forms a cytoplasmic gate by hydrogen bonding with the backbone carbonyl of W373 and the hydroxyl of S377. (D) Tripartite interactions between TM1, TM5, and ICH1. This view is rotated 190° as indicated for clarity. (E) Cylinder representation of the N- and C-terminal domains separated and rotated 90° (left) and 90° (right) show the extent of structural change between inward-open (green) and outward-occluded (yellow) states. With substrate bound, TM7 bends approximately 20° to partially occlude the substrate from the periplasmic side. The helical discontinuity of TM4 in the outward-occluded state and of TM10 in the inward-open state are indicated by black arrows. Arg47, arginine-47; DgoT, D-galactonate transporter; Fo-Fc, difference map; ICH, intracellular helix; TM, transmembrane.