Metal Substrate Translocation and Transport Mechanism of the Sinorhizobium melilotiP1B‐5‐type ATPase Nia Revealed by In‐vitro Transport Assays in Proteoliposomes

Abstract

P1B‐type ATPases are a class of transmembrane transporter proteins responsible for metal substrate translocation, and export across cellular membranes. These ion pumps are classified into seven sub‐families (P1B‐1‐P1B‐7‐type) based on conserved amino acid motifs in their transmembrane helices that appear to control each sub‐family’s substrate selectivity. The P1B‐5‐type sub‐family is widely found in nitrogen fixing bacteria, such as Sinorhizobium meliloti,which form a unique symbiotic relationship with legumes. This symbiotic relationship is highly dependent on the nutrition trafficking between the two species, including a tight control of transition metal homeostasis. The P1B‐5‐type ATPase Nia from S. Meliloti is recognized as a putative iron and nickel transporter, and besides possessing the typical P‐type ATPase domains and topology, it features of a C‐terminal hemerythrin‐like domain containing a di‐iron binding site involved in oxygen sensing, as proposed by in‐vivo studies. However, the substrate transport across lipid bi‐layers, the overall mechanism for cargo translocation and the transport kinetic parameters remain elusive due to lack of molecular tools to study putative substrate transport across membranes in real‐time.In this study, we coupled metal‐stimulated ATPase activity assays with an experimental platform based on multiple fluorescence sensor probes, to study substrate transport mechanism and translocation kinetics in real‐time with Nia reconstituted in a native‐like environment. We expressed and purified Nia in detergent micelles and subsequently incorporated it in artificial small unilamellar lipid bi‐layer vesicles (proteoliposomes) which allow to study the substrate specificity and transport mechanism in real‐time. The proteoliposomes were used as an in‐vitrotool to determine the kinetic parameters for metal transport by encapsulating fluorescent detector probes featuring turn‐on florescence signal upon substrate ion binding and translocation.The study revealed that Fe2+, Ni2+ and Co2+are actively translocated by Nia and the kinetic parameter analysis demonstrated that Fe2+ is the preferential Nia substrate. In parallel, by encapsulating in the proteoliposome lumen pH and membrane sensitive fluorescent probes we are also revealing the chemistry underlying the mechanism of transport and potential ions involved as counter‐transported cargos in P1B‐5‐type ATPase catalytic cycle, with the aim of revealing unique molecular features for this subclass of transmembrane metal pumps.