Structural basis of K<+>/H<+> antiport in YcgO and its inhibition by unphosphorylated PtsN

Cation-proton antiporters (CPAs) are vital for the maintenance of ionic homeostasis and normal physiology among diverse cell types. Despite recent insights into K<+>/H<+> exchange transporters, the diversity in their structural organization and regulatory mechanisms of K<+>-specific CPAs are minimally understood. Here, we explore the architecture of an E. coli CPA1 K<+>/H<+> antiporter, YcgO and its inhibition by the unphosphorylated form of PtsN, the terminal protein of a regulatory phosphorelay, using cryoEM structures at 3.4 [A] and 3.2 [A] resolution, respectively. Homodimeric YcgO bound to K<+> ions in the occluded conformation, harbors additional linked cytosolic domains, RCK and CorC, to regulate the movement of the transport helices within the YcgO dimer. These domains are the sites of interaction and efflux inhibition by unphosphorylated PtsN, which interacts with the CorC domains with high affinity and allosterically augments inhibitory interactions of CorC with transport helices of YcgO. Inhibition is relieved leading to constitutive activation, upon disrupting the CorC-transport conduit interface. This study illuminates the structural basis of K<+> efflux mediated through regulation of a K<+>/H<+> antiporter in E. coli and related prokaryotes via a metabolic network involving a regulatory phosphorelay.