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bioRxiv · 10.1101/2025.03.04.641403

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

Madapally, H. V.·Hussein, A. K.·Eriksen, M. W.·Pedersen, B. P.·Khandelia, H.·Stokes, D.
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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.

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Madapally, H. V., Hussein, A. K., Eriksen, M. W., Pedersen, B. P., Khandelia, H., Stokes, D.. 2025-03-11. On the mechanism of K+ transport through the inter-subunit tunnel of KdpFABC. https://doi.org/10.1101/2025.03.04.641403

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