Sodium recognition by the Na+/Ca2+ exchanger in the outward-facing conformation

Na⁺/Ca²⁺ exchangers (NCXs) are ubiquitous membrane transporters with a key role in Ca²⁺ homeostasis and signaling. NCXs mediate the bidirectional translocation of either Na⁺ or Ca²⁺, and thus can catalyze uphill Ca²⁺ 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⁺/Ca²⁺ and Ca²⁺/Ca²⁺ exchange by wild-type and mutagenized NCX_Mj confirm that transport of both Na⁺ and Ca²⁺ 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 Ca²⁺ is not. Altogether, these findings provide the basis for further experimental and computational studies of the conformational mechanism of this exchanger.Ca²⁺ signals control a variety of cellular processes essential for the basic function of multiple organs. In cardiac cells, for example, Ca²⁺ release from the sarcoplasmic reticulum is a necessary step for heart contraction, whereas Ca²⁺ extrusion from the cell is required for cardiac relaxation. These fluctuations in the cytosolic Ca²⁺ concentration underlie the initiation of the heartbeat (1, 2). Na⁺/Ca²⁺ exchangers (NCXs) play a central role in the homeostasis of cellular Ca²⁺ (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 Ca²⁺ or Na⁺ across the membrane; thus, they can harness a transmembrane sodium motive force to energize Ca²⁺ transport against a concentration gradient. For example, the cardiac exchanger NCX1 mediates the extrusion of intracellular Ca²⁺ driven by a Na⁺ transmembrane gradient maintained by the Na⁺/K⁺ ATPase (3, 6).Numerous electrophysiological studies over the past three decades have analyzed the functional and regulatory properties of these important exchangers. It is well established that NCXs are reversible and electrogenic, but it has been debated whether the Na⁺/Ca²⁺ exchange stoichiometry is 3:1 or 4:1 (3, 5, 7–12). In any case, NCXs can also facilitate Na⁺/Na⁺ and Ca²⁺/Ca²⁺ exchange, implying that the translocation of Na⁺ and Ca²⁺ are two distinct reactions (3, 5, 6, 13). NCXs are regulated by several factors, such as cytosolic Na⁺ and Ca²⁺ concentrations, pH, ATP, and PIP₂ (5, 6). Ca²⁺ regulation in particular involves accessory cytoplasmic domains not directly implicated in the ion-exchange function of the transmembrane domain (5, 14, 15). It is in highly conserved regions within the latter domain, known as α1 and α2 (in transmembrane helices TM2/TM3 and TM7/TM8, respectively), where specific polar and carboxylic amino acids have been identified to be crucial for ion binding and transport (6, 16–18). Similar sequence motifs are found in related antiporters that exchange Na⁺ for K⁺ and Ca²⁺ (NCKX), and Ca²⁺ for H⁺ (CAX) (19–21).In a recent breakthrough, the NCX exchanger from the archaeobacterium Methanocaldococcus jannaschii was isolated and functionally reconstituted, and its crystal structure determined at 1.9-Å resolution (22). The structure of NCX_Mj revealed an intriguing architecture consisting of two inverted topological repeats of five transmembrane helices each (Fig. 1A), which adopt similar but not identical conformations (Fig. S1). This structural asymmetry is likely to underlie the ability of the exchanger to adopt outward- and inward-open conformations alternately, as is mandatory in secondary-transport processes (23, 24). Importantly, the electron density map for NCX_Mj also revealed clear peaks for four putative ion-binding sites in the α1 and α2 regions, involving several of the residues previously identified as essential (Fig. 1B). One of these sites, referred to as S_Ca, also produced an anomalous scattering signal, somewhat weak but indicative of the presence of a bound Ca²⁺. The sites referred to as S_ext and S_int appeared to be Na⁺ sites, based on chemical and geometric considerations. The fourth site, S_mid, was tentatively interpreted also as a Na⁺ site, although in this case the ion-coordination number and coordination geometry would be atypical (22).Open in a separate windowFig. 1.Structure of the Na⁺/Ca²⁺ exchanger from M. jannaschii in an outward-facing conformation. (A) The architecture of NCX_Mj (PDB entry 3V5U) consists of two repeats of five transmembrane helices each (orange, marine cartoons), which adopt opposite orientations in the membrane (the cytoplasmic side is below). Four putative ion-binding sites (indicated by gray spheres) are found halfway across the transmembrane region, flanked by helices TM2–TM3 and TM7–TM8. The structures of the five-helix repeats are highly similar but not identical (Fig. S1). The major differences are the orientation of TM1/TM6 relative to the rest of the repeat, and a bend in the periplasmic half of TM7, not observed in the equivalent region of TM2. These structural asymmetries likely underlie the ability of the exchanger to alternatively adopt outward- and inward-facing conformations (24). (B) Close-up of the four putative ion-binding sites. The side-chain and backbone groups lining these sites are highlighted; the hypothetical ion coordination contacts are indicated in each case. (C) All-atom simulation model of NCX_Mj embedded in a phospholipid membrane. The membrane consists of 208 POPC molecules, and protein and membrane are hydrated by ～14,900 water molecules. Chloride ions (omitted for clarity) were also included to counter the net charge of the ion–protein complex. The overall system size is ～77,300 atoms. (D) Ion configuration that is most likely to correspond to the crystal structure of NCX_Mj, according to the structural and thermodynamic analyses described in Figs. 2 and ?and3.3. A water molecule (W) occupies the S_mid site, while Na⁺ ions occupy S_ext, S_Ca, and S_int. Note that D240 is protonated, and that the side-chain carboxamide group of N81 is inverted relative to the original assignment. The figure shows the conformation that is most frequently observed in a 200-ns MD simulation, according to an RMSD-based clustering analysis comprising the residues highlighted in B.If one assumes that the exchange of Na⁺ and Ca²⁺ mediated by NCX_Mj proceeds according to a “ping-pong” mechanism, in analogy with other NCXs (13), binding of Na⁺ and Ca²⁺ ought to be mutually exclusive (22), even if the exchange stoichiometry is 3:1 rather than 4:1. Thus, the detection of concurrent electron density signals for three Na⁺ and one Ca²⁺ must reflect the coexistence of protein molecules with either of these two ion configurations in the crystal lattice. On the other hand, configurations in which Na⁺ and Ca²⁺ are simultaneously bound cannot be entirely ruled out; indeed, a minor transport mode has been reportedly observed for cardiac NCX1 whereby one Na⁺ and one Ca²⁺ are cotranslocated (9). Either way, it is puzzling that the local structure of the binding sites revealed by the electron density is uniquely defined, as it seems unlikely that this structure is identical irrespective of which ion type is bound. This lack of disorder could be explained if only one of the ion configurations that hypothetically coexist in the protein crystal was significantly populated, but it is not immediately apparent which of these configurations is represented by the data. Thus, notwithstanding the groundbreaking insights provided by the NCX_Mj structure, the interpretation of the electron density in regard to the ion configuration is, in our view, not straightforward: could the proposed Na⁺ occupancy of the suboptimal S_mid site result from the concurrent binding of Ca²⁺, explaining the minor transport mode observed for NCX1? If so, would S_mid remain occupied by Na⁺ in the absence of Ca²⁺? Or is the S_Ca site alternatively occupied by Na⁺ and Ca²⁺? If so, does the electron density signal detected in S_mid reflect a fourth Na⁺ ion? Or could it be a water molecule, or another cation, such as K⁺? It is worth noting that the amino acid composition of the binding sites in NCX_Mj is more akin to that of NCKX exchangers, which cotransport K⁺ along with Ca²⁺, than to NCX homologs (Fig. S2), including those of Escherichia coli and Methanosarcina acetivorans (25). The functional reconstitution of NCX_Mj showed that Na⁺/Ca²⁺ exchange in NCX_Mj is not influenced by a K⁺ gradient (22), but it is conceivable that K⁺ is constitutively bound throughout the transport cycle.Here, we use all-atom molecular-dynamics (MD) simulations and free-energy calculations of NCX_Mj to systematically assess a series of possible ion configurations, both in terms of their consistency with the protein conformation observed in the experimental crystal structure, and from a thermodynamic standpoint. This computational analysis yields a clear-cut testable hypothesis, which we examine experimentally via Na⁺/Ca²⁺ and Ca²⁺/Ca²⁺ exchange assays for wild-type and mutagenized NCX_Mj.