FIGURE 2.

Structure 2, NADPH-binding site in SmTGRfl. Panel A, zoom in the FAD active site. The NADPH and FAD are shown as green sticks; Tyr²⁹⁶, Ser²⁹⁵, Cys¹⁵⁴, and Cys¹⁵⁹ are shown as red sticks, and His⁵⁷¹ of the partner subunit is shown as blue sticks. Upon NADPH binding, the loop 295–297 changes conformation re-orienting the side chains of Ser²⁹⁵ and Tyr²⁹⁶ with respect to Structure 1 to make room for the reductant. The Cys¹⁵⁹–Cys¹⁵⁴ couple is partially reduced, and accordingly, Cys¹⁵⁴ is found in a double conformation. In the reduced conformation (50% occupancy), the sulfur of Cys¹⁵⁴ points toward the solvent and is in contact with a water molecule (2.7 Å, shown as red ball), which is kept in place by His⁵⁷¹. Panel B, GSH-binding site on TR domain of Structure 2. The GSH (green sticks) is found in a position that in other TGR structures is usually occupied by the loop 397–404 (cyan ribbon). The ligand is found in a pocket formed by one α-helix (Leu³⁹⁷–Thr⁴⁰⁴), a β-strand (Lys²²⁷–Leu²³⁰), and the nucleotide moiety of the FAD. GSH makes a mixed disulfide with Cys⁴⁰² and forces the loop 397–404 to adopt a α-helical secondary structure (red ribbon) by turning around two pivot residues Leu³⁹⁷ and Thr⁴⁰⁴ (shown as full black circles). One of the gates of the NADPH-binding site (full black square) is four residues upstream of Leu³⁹⁷.