Structure and mechanism of the K+/H+ exchanger KefC

Abstract Intracellular potassium (K+) homeostasis is fundamental to cell viability. In addition to channels, cellular K+ levels are maintained by various ion-transporters. One major family are the proton-driven K+ efflux transporters. In gram-negative bacteria, K+/H+ exchange activity is important for osmoregulation and, in plants, their activity is critical for efficient photosynthesis and growth. Despite their wide-spread importance, the structure and molecular basis for K+-selectivity is poorly understood. Here, we report the cryo-EM structure of the model glutathione (GSH)-gated K+ efflux transporter KefC from E. coli at 3.2 Å resolution. KefC forms a homodimer and is overall similar to the Na+/H+ antiporter NapA captured in an inward-facing conformation. By structural assignment of a coordinated K+ ion, MD simulations and SSM-based electrophysiology, we demonstrate how the ion-binding site in KefC is adapted for binding a dehydrated K+ ion, which contrasts with NapA, which coordinates a partially hydrated Na+ ion. The substrate-K+ ion is completely enclosed within the transport domains, consistent with an elevator alternating-access mechanism. KefC harbours C-terminal RCK regulatory domains, similar to the TrkA domain of K+-channels, forming homodimers that are stabilised by AMP and GSH. In the current KefC structure, a pair of domain-swapped helices in the RCK domains inhibit transport by directly interacting with the ion-transport domains. Taken together, we propose that KefC is activated by detachment of the RCK domains and ion-selectivity exploits the biophysical properties likewise adapted by cation ion-channels.