Conformational free-energy landscapes of a Na+/Ca2+exchanger explain its alternating-access mechanism and functional specificity

Secondary-active transporters catalyze the movement of myriad substances across all cellular membranes, typically against opposing concentration gradients, and without consuming any ATP. To do so, these proteins employ an intriguing structural mechanism evolved to be activated only upon recognition or release of the transported species. We examine this self-regulated mechanism using a homolog of the cardiac Na + /Ca 2+ exchanger as a model system. Using advanced computer simulations, we map out the complete functional cycle of this transporter, including unknown conformations that we validate against existing experimental data. Calculated free-energy landscapes reveal why this transporter functions as an antiporter rather than a symporter, why it specifically exchanges Na + and Ca 2+ , and why the stoichiometry of this exchange is exactly 3:1. We also rationalize why the protein does not exchange H + for either Ca 2+ or Na + , despite being able to bind H + and its high similarity with H + /Ca 2+ exchangers. Interestingly, the nature of this transporter is not explained by its primary structural states, known as inward- and outward-open conformations; instead, the defining factor is the feasibility of conformational intermediates between those states, wherein access pathways leading to the substrate binding sites become simultaneously occluded from both sides of the membrane. This analysis offers a physically-coherent, broadly transferable route to understand the emergence of function from structure among secondary-active membrane transporters. Significance The class of membrane proteins known as secondary-active transporters mediate a wide range of critical cellular processes, including nutrient uptake, transmembrane signaling, and resistance to cytotoxic compounds, like human-made drugs. A detailed understanding of their molecular mechanisms is therefore of interest not only from a fundamental standpoint, but also because it will facilitate the design of inhibitors or stimulators that may be used as therapeutic agents. This study provides a conceptual mechanistic framework, grounded on statistical thermodynamics, that bridges the specific physiological function of these proteins and their molecular structure. While the study is focused on a particular subclass of transporters involved in cardiac physiology and cellular Ca 2+ homeostasis, we envisage our conclusions will be broadly applicable.