How the Substrate Occupancy of a Membrane Transporter Determines the Viability of its Alternating-Access Mechanism and thus its Functional Specificity

Abstract

Na+/Ca2+ exchangers (NCX) are important membrane antiporters involved in Ca2+ homeostasis. In typical physiological conditions, their role is to extrude cytosolic Ca2+ in exchange for extracellular Na+. Here, we assess the mechanisms of ion recognition and exchange of a prokaryotic NCX homolog using conformational free-energy landscapes of outward- and inward-facing conformations of the transporter bound to Na+ or Ca2+ in various occupancies as well as in an apo state. These calculations, based on advanced molecular dynamics simulations, elucidate the factors that control the alternating-access mechanism of this antiporter. The inward-facing conformation, not known experimentally, was independently deduced from enhanced sampling molecular simulations and repeat-swap molecular modeling. Our results reveal that only specific ion occupancies, namely either 3 Na+ or 1 Ca2+, permit the transporter to access an occluded state that is intermediate between the outward- and inward-facing state. By contrast, the apo state or other ion occupancies lock the protein into either the outward- or inward-facing conformations, thereby stalling the transport mechanism. Our analysis indicates that the feasibility of the occluded state, and therefore the alternating-access transition, is determined by the precise balance of ion-protein and water-protein interactions, which is strongly dependent on the occupancy state of the protein. In summary, this study provides a molecular-level explanation for the observation that NCX operate through a strictly-coupled “ping-pong” mechanism, and for their ion-exchange stoichiometry, namely 3Na+:1Ca2+. More broadly, our analysis suggests that the functional specificity of membrane transporters featuring an alternating-access mechanism, i.e. whether they function as symporters, antiporters or uniporters, and their precise stoichiometry, is in large part explained by the feasibility of the conformational transitions that result in occlusion of the substrate-binding sites to both sides of the membrane.