Mechanistic Dissection of Cu(I)‐translocating P‐type ATPase Pumps by I n‐vitro Real‐Time Transport in Artificial Lipid Bilayer Vesicles

Abstract

Copper is an essential element in biological systems where it plays key roles as a co-factor in diverse cuproenzymes and in signaling processes. Cu(I) transporting P-type ATPases (CopA) are primary active transmembrane transporter proteins that utilize the energy of ATP hydrolysis to catalyze the extrusion of Cu(I) ions across cell membranes. Their activity plays a gatekeeper role in controlling intracellular Cu(I) levels and homeostasis, a key process for survival in all kingdoms of life. In humans, mutations in these Cu(I) pumps lead to the Menkes syndrome and the Wilson's disease. In pathogenic bacteria CopA proteins can act as virulence factors to overcome the Cu(I) stress generated by the host defense system, thereby representing a potential therapeutic target against pathogens causing various fatal diseases. Even though the structure and function of these transporters has been established, the overall mechanism for substrate translocation, their transport kinetic parameters and electrogenicity remain elusive. In this work we developed a novel coupled experimental platform, centered on multiple fluorescence reporter probes that permits to study substrate, the transport mechanism and translocation kinetics in real-time in Cu(I)-pumps reconstituted in a native-like environment. We reconstituted the model Cu(I)-pump from E. coli (EcCopA) and its inactive mutants in artificial small unilamellar vesicles (proteoliposomes). We quantitatively characterized the chemistry underlying substrate transport, putative counterions and charge translocation by encapsulating in the liposome lumen fluorescence detector probes (CTAP-3, Pyranine and Oxonol VI) responsive to diverse stimuli (Cu(I) substrate, pH and transmembrane potential). The study established that Cu(I) pumps are primary-active uniporters and electrogenic pumps that build a transmembrane potential by translocating one positive charge (Cu(I)) per ATP hydrolysis cycle without coupling it to counterion transport. This analysis further reveals mechanistic differences in substrate translocation by CopA proteins in comparison to other characterized P-type ATPase classes. Moreover, the developed approach can be extended as a general biophysical tool to investigate other P-type ATPase pumps in which the mechanism of substrate translocation has not yet been established.