On the mechanism of K+transport through the inter-subunit tunnel of KdpFABC

ABSTRACT KdpFABC is an ATP-dependent membrane complex that enables prokaryotes to maintain potassium homeostasis under potassium-limited conditions. It features a unique hybrid mechanism combining a channel-like selectivity filter in KdpA with the ATP-driven transport functionality of KdpB. A key unresolved question is whether K + ions translocate through the inter-subunit tunnel as a queue of ions or within a hydrated environment. Using molecular dynamics (MD) simulations, metadynamics, anomalous X-ray scattering, and biochemical assays, we demonstrate that the tunnel is predominantly occupied by water molecules rather than multiple K + ions. Our results identify only one stable intermediate binding site for K + within the tunnel, apart from the canonical sites in KdpA and KdpB. Free energy calculations reveal a substantial barrier (∼22 kcal/mol) at the KdpA-KdpB interface, making spontaneous K + translocation unlikely. Furthermore, mutagenesis and functional assays confirm previous findings that Phe232 at this interface plays a key role in coupling ATP hydrolysis to K + transport. These findings challenge previous models proposing a continuous wire of K + ions through the tunnel and suggest the existence of an as-yet unidentified intermediate state or mechanistic detail that facilitates K + movement into KdpB.