pH-Dependent Conformational Transition of the Glutamate-GABA Antiporter GadC Revealed by ¹⁹F NMR

The glutamate-γ-aminobutyric acid (Glu-GABA) antiporter GadC is the key element of the acid resistance systems, which enables food-borne hemorrhagic Escherichia coli to endure the highly acidic conditions of the stomach (pH ∼2). Although the crystal structure of GadC in its inactive state at pH 8.0 has been determined, the structure and the molecular dynamics underlying the transition between active and inactive states have not been elucidated. Here, we employed site-specific incorporation of unnatural amino acids and ¹⁹F-detected NMR spectroscopy to dissect the conformational plasticity of the core domain, gate domain, and C-plug of GadC in response to pH. Our results revealed that each of these domains undergoes significant structural rearrangement upon a decrease in pH. Specifically, both the core and gate domains adopt faster conformation exchange under acidic conditions, which are essential for facilitating substrate transport. Meanwhile, the C-plug is found in both active and inactive forms at alkaline pH. As the pH decreases, the inactive conformation of the C-plug transitions entirely to an active state, highlighting its critical role in pH-dependent transport regulation and acid resistance. These findings reveal a dynamic mechanism in pH-dependent activation of GadC, thereby providing a molecular framework for GadC-mediated acid resistance in the gastrointestinal tract.