Plastic recognition and electrogenic uniport translocation of 1(st)-, 2(nd)-, and 3(rd)-row transition and post-transition metals by primary-active transmembrane P(1B-2)-type ATPase pumps

Transmembrane P(1B)-type ATPase pumps catalyze the extrusion of transition metal ions across cellular lipid membranes to maintain essential cellular metal homeostasis and detoxify toxic metals. Zn(ii)-pumps of the P(1B-2)-type subclass, in addition to Zn(2+), select diverse metals (Pb(2+), Cd(2+) and Hg(2+)) at their transmembrane binding site and feature promiscuous metal-dependent ATP hydrolysis in the presence of these metals. Yet, a comprehensive understanding of the transport of these metals, their relative translocation rates, and transport mechanism remain elusive. 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 metal substrates. Promiscuous coordination plasticity, guarantees diverse, yet defined, cargo selectivity coupled to their translocation.