Keeping Secondary Transporters under Control: Lessons from a Na+/Ca2+ Exchanger

Abstract

Secondary-active transporters catalyze the translocation of substances across biological membranes, driven by the electrochemical gradient of one of the transported species, typically Na+ or H+. The mechanism of these transporters has been widely rationalized as a conformational cycle (alternating-access model) that exposes substrate binding sites within the protein to one or the other side of the membrane (or none), but not both simultaneously. The electrochemical gradient drives the cycle in one direction but is not required to interconvert between different conformational states (as opposed to voltage-gated ion-channels). However, active transport does require that the interconversion between outward and inward facing states occur only upon binding of specific substrates, and in uniquely defined stoichiometries. The physical basis of this mechanism of conformational control, however, remains to be established. Here, we address this central question for a prokaryotic homolog of the cardiac Na+/Ca2+ exchanger, NCX_Mj, which transports either three Na+ or one Ca2+ across the membrane. Specifically, we determine how Na+/Ca2+ recognition by the transporter outward-facing state reshapes its conformational free-energy landscape, which we examine via enhanced-sampling molecular-dynamics simulations and crystallographic titration experiments. Our results demonstrate that only upon binding of three Na+ or one Ca2+ can the protein adopt a state occluded to both sides of the membrane, which necessarily precedes the transition to the inward-facing conformation. Binding sites depletion and/or H+ binding, by contrast, eradicates the occluded state population, and induces the opening of hydrated access pathways connecting these sites to the surrounding solution. Interestingly, water in these pathways is markedly polarized, in part explaining the large energetic cost associated with occlusion. This study provides clear evidence that it is by inducing or precluding the formation of occluded, dehydrated states that substrate recognition controls the alternating-access transition in secondary transporters.