Transition metal transporting P-type ATPases: terminal metal-binding domains serve as sensors for autoinhibitory tails.

Copper is an essential micronutrient and yet is highly toxic to cells at elevated concentrations. P-ATPase proteins are critical for this regulation, providing active extrusion across cellular membranes. One unique molecular adaptation of P-ATPases compared to other P-type ATPases is the presence of metal-binding domains (MBDs) at the cytosolic termini, which however are poorly characterized with an elusive mechanistic role. Here we present the MBD architecture in metal-free and metal-bound forms of the archetype Cu-specific P-ATPase LpCopA, determined using NMR. The MBD is composed of a flexible tail and a structured core with a metal ion binding site defined by three sulfur atoms, one of which is pertinent to the so-called CXXC motif. Furthermore, we demonstrate that the MBD rather than being involved in ion delivery likely serves a regulatory role, which is dependent on the classical P-type ATPase E1-E2 transport mechanism. Specifically, the flexible tail appears responsible for autoinhibition while the metal-binding core is used for copper sensing. This model is validated by a conformation-sensitive and MBD-targeting nanobody that can structurally and functionally replace the flexible tail. We propose that autoinhibition of Cu-ATPases occurs at low copper conditions via MBD-mediated interference with the soluble domains of the ATPase core and that metal transport is enabled when copper levels rise, through metal-induced dissociation of the MBD. This allows P-ATPase 'vacuum cleaners' to tune their own activity, balancing the levels of critical micronutrients in the cells.