Na + /Ca 2+ exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca 2+ homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na + or Ca 2+ , and thus can catalyze uphill Ca 2+ transport driven by a Na + gradient, or vice versa. In a major breakthrough, a prokaryotic NCX homolog (NCX_Mj) was recently isolated and its crystal structure determined at atomic resolution. The structure revealed an intriguing architecture consisting of two inverted-topology repeats, each comprising five transmembrane helices. These repeats adopt asymmetric conformations, yielding an outward-facing occluded state. The crystal structure also revealed four putative ion-binding sites, but the occupancy and specificity thereof could not be conclusively established. Here, we use molecular-dynamics simulations and free-energy calculations to identify the ion configuration that best corresponds to the crystallographic data and that is also thermodynamically optimal. In this most probable configuration, three Na + ions occupy the so-called S ext , S Ca , and S int sites, whereas the S mid site is occupied by one water molecule and one H + , which protonates an adjacent aspartate side chain (D240). Experimental measurements of Na + /Ca 2+ and Ca 2+ /Ca 2+ exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na + and Ca 2+ requires protonation of D240, and that this side chain does not coordinate either ion at S mid . These results imply that the ion exchange stoichiometry of NCX_Mj is 3:1 and that translocation of Na + across the membrane is electrogenic, whereas transport of Ca 2+ is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.secondary transporters | membrane antiporters | ion specificity | CaCA superfamily | molecular-dynamics simulations C a 2+ signals control a variety of cellular processes essential for the basic function of multiple organs. In cardiac cells, for example, Ca 2+ release from the sarcoplasmic reticulum is a necessary step for heart contraction, whereas Ca 2+ extrusion from the cell is required for cardiac relaxation. These fluctuations in the cytosolic Ca 2+ concentration underlie the initiation of the heartbeat (1, 2). Na + /Ca 2+ exchangers (NCXs) play a central role in the homeostasis of cellular Ca 2+ (3-5). These integral membrane proteins are ubiquitous in many types of tissues including the heart, brain, and kidney (4), and consequently their dysfunction is associated with numerous human pathologies such as cardiac arrhythmia, hypertension, skeletal muscle dystrophy, and postischemic brain damage (5). NCXs facilitate the translocation of either Ca 2+ or Na + across the membrane; thus, they can harness a transmembrane sodium motive force to energize Ca 2+ transport against a concentration gradient. For example, the cardiac exchanger NCX1 mediates the extrusion of intracellular Ca 2+ driven by a Na + transmembrane gradient maintained by the Na ...

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In analogy with many other proteins, Na+/Ca2+ exchangers (NCX) adapt an inverted twofold symmetry of repeated structural elements, while exhibiting a functional asymmetry by stabilizing an outward-facing conformation. Here, structure-based mutant analyses of the Methanococcus jannaschii Na+/Ca2+ exchanger (NCX_Mj) were performed in conjunction with HDX-MS (hydrogen/deuterium exchange mass spectrometry) to identify the structure-dynamic determinants of functional asymmetry. HDX-MS identified hallmark differences in backbone dynamics at ion-coordinating residues of apo-NCX_Mj, whereas Na+or Ca2+ binding to the respective sites induced relatively small, but specific, changes in backbone dynamics. Mutant analysis identified ion-coordinating residues affecting the catalytic capacity (kcat/Km), but not the stability of the outward-facing conformation. In contrast, distinct “noncatalytic” residues (adjacent to the ion-coordinating residues) control the stability of the outward-facing conformation, but not the catalytic capacity. The helix-breaking signature sequences (GTSLPE) on the α1 and α2 repeats (at the ion-binding core) differ in their folding/unfolding dynamics, while providing asymmetric contributions to transport activities. The present data strongly support the idea that asymmetric preorganization of the ligand-free ion-pocket predefines catalytic reorganization of ion-bound residues, where secondary interactions with adjacent residues couple the alternating access. These findings provide a structure-dynamic basis for ion-coupled alternating access in NCX and similar proteins.

Na+/Ca2+ exchanger (NCX) proteins mediate Ca2+-fluxes across the cell membrane to maintain Ca2+ homeostasis in many cell types. Eukaryotic NCX contains Ca2+-binding regulatory domains, CBD1 and CBD2. Ca2+ binding to a primary sensor (Ca3-Ca4 sites) on CBD1 activates mammalian NCXs, whereas CALX, a Drosophila NCX ortholog, displays an inhibitory response to regulatory Ca2+. To further elucidate the underlying regulatory mechanisms, we determined the 2.7 Å crystal structure of mammalian CBD12-E454K, a two-domain construct that retains wild-type properties. In conjunction with stopped-flow kinetics and SAXS (small-angle X-ray scattering) analyses of CBD12 mutants, we show that Ca2+ binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca2+-driven interdomain switch controls slow dissociation of “occluded” Ca2+ from the primary sensor and thus dictates Ca2+ sensing dynamics. In the Ca2+-bound conformation, the interdomain angle of CBD12 is very similar in NCX and CALX, meaning that the interdomain distances cannot account for regulatory diversity in NCX and CALX. Since the two-domain interface is nearly identical among eukaryotic NCXs, including CALX, we suggest that the Ca2+-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.

The crystal structures of the CBD1 and CBD2 domains of the Na ؉ /Ca 2؉ exchanger protein (NCX1) provided a major breakthrough in Ca 2؉ -dependent regulation of NCX1, although the dynamic aspects of the underlying molecular mechanisms are still not clear. Here we provide new experimental approaches for evaluating the kinetic and equilibrium properties of Ca 2؉ interaction with regulatory sites by using purified preparations of CBD1, CBD2, and CBD12 proteins. CBD12 binds ϳ6 Ca 2؉ ions (mol/mol), whereas the binding of only ϳ2 Ca 2؉ ions is observed (with a Hill coefficient of n H ‫؍‬ ϳ2) either for CBD1 or CBD2. In the absence of Mg 2؉ , CBD1 has a much higher affinity for Ca