ATP-driven P-type ATPase ion pumps are key systems for the selective translocation of essential and toxic transition metal ions across cell membranes in all the domains of life. Their activity plays a gatekeeper role in controlling metal concentrations within cells to ensure correct metal homeostasis and active extrusion of toxic metals.
Zn(II)-pumps of the P_1B-2-type subclass, in addition to Zn²⁺, select diverse metals (Pb²⁺, Cd²⁺ and Hg²⁺) at their transmembrane binding site and feature promiscuous metal-dependent ATP hydrolysis in the presence of these metals. We developed a platform for the characterization of primary-active Zn(II)-pumps in proteoliposomes to study metal selectivity, translocation events and transport mechanism in real-time, employing a “multi-probe” approach with fluorescent sensors responsive to diverse stimuli (metals, pH and membrane potential). Together with atomic-resolution investigation of cargo selection by X-ray Absorption Spectroscopy (XAS), we demonstrate that Zn(II)-pumps are electrogenic uniporters that preserve the transport mechanism with 1^st-, 2^nd- and 3^rd-row transition and post-transition metal substrates. Promiscuous coordination plasticity, guarantees diverse, yet defined, cargo selectivity coupled to their translocation.
Our work reveals how the strict interplay between the nature of the coordinating ligands, the geometry of metal binding in the transmembrane helices, and the unique coordination chemistry determines substrate selection and translocation. Overall, we provide new highlights on the diverse mechanism of transport in P-type metal pumps and address how the energy stored in ATP is transduced into the vectorial flux of metals across lipid bilayers.

Priyanka Basak

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