(A) The cross-linked D417C backbone (PDB 5HD8, green) superposes with WT (PDB 1OTS, blue) (RMSD 0.57 Å for 862 Cα atoms). (B) Extra density between residues 417 on the two subunits was modeled as a disulfide bridge, shown in stereo. (C) Close up stereo view of key residues around the Cl- (upper panel) and H+ (lower panel) permeation pathways. In the upper panel, the residues shown (E148, S107, and Y445) are the same as those depicted in Figure 1A. In the lower panel, also shown are E203, the internal H+-transfer site (Accardi et al., 2005) and A404, a residue lining the portal for H+ entry from the intracellular solution (Han et al., 2014). Cl- modeled in the central binding site is depicted as green and blue spheres. 2F0-Fc maps are contoured at 1σ. (D) ITC experiments show Cl- binding to WT, D417C, and D417C cross-linked with 100 µM CuP. Top panels: heat liberated when 20 mM KCl is titrated into the ITC cell containing 25–50 µM protein (WT, 25 µM; D417C, 50 µM; D417C+CuP, 30 µM). (E) Summary data for ITC experiments, ± SEM. WT, n=3 from two separate protein preparations; D417C, n=4 from four separate preparations; cross-linked D417C n=4 from three separate protein preparations.