Fig. 7.

Interactions between I239T/G354E and DXC. a Close-up view of the DXC-binding site within I239T/G354E, with the N and C domains colored cyan and yellow, respectively. The bound DXC (green) and relevant amino acids are shown in stick models, and the close-range interactions are highlighted by dashed lines. b Overlay of the DXC-bound Q131R (light blue, PDB 4ZP0) and I239T/G354E, with the DXC-binding amino acids and DXC drawn as stick models. The Q131R-bound DXC is colored light pink and the C1-OH groups are highlighted by black arrows. c Fluorescence measurement of a solution containing 4 µM MdfA (green), revealing its ability to release one proton per protein molecule upon DXC binding. As a comparison, the addition of DXC to a solution containing 4 µM I239T/G354E (red), failed to trigger the release of H⁺. d DXC/H⁺ antiport observed in the everted membrane vesicles expressing MdfA (green). H⁺ movement was monitored by measurement of acridine orange fluorescence, which is shown in arbitrary units (a.u.). By contrast, DXC failed to trigger H⁺ movement in the everted membrane vesicles harboring I239T/G354E (red) or vector (black). The traces are representative of experiments performed in triplicate using two different preparations of everted membrane vesicles