Sodium recognition by the Na ⁺ /Ca ²⁺ exchanger in the outward-facing conformation

Abstract

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 ...

Structure-Functional Basis of Ion Transport in Sodium–Calcium Exchanger (NCX) Proteins

“…However, only recently has a major advancement in understanding the structural basis for this mechanism been achieved by solving the crystal structure of NCX_Mj from the arachaeabacterium Methanococcus jannaschii [24]. In combination with known crystal structures, specially designed MD simulations and extended kinetic analyses of ion-fluxes in mutants revealed that the simultaneous occupation of 3Na + and 1Ca 2+ is thermodynamically forbidden [25,26,27]. Similarly to mammalian NCX, the NCX_Mj protein also translocates Na + and Ca 2+ with a stoichiometry of 3:1, although in contrast with NCX_Mj, the three mammalian genes NCX1-3 contain a large cytosolic regulatory loop (~520 aa) between helices 5 and 6 [28,29,30].…”

“…To resolve the uncertainty regarding the specificity and occupancy of the binding sites, especially S mid , molecular dynamics simulations were performed to systematically assess the different possible ion configurations [25]. This systematic analysis suggested that 3Na + occupy S int , S Ca , and S ext or that 1Ca 2+ occupies S Ca , whereas in the Na + -bound state, D240 is protonated and a water molecule occupies S mid [25].…”

“…This systematic analysis suggested that 3Na + occupy S int , S Ca , and S ext or that 1Ca 2+ occupies S Ca , whereas in the Na + -bound state, D240 is protonated and a water molecule occupies S mid [25]. This interpretation was verified experimentally by ion-flux assays of the D240N mutant, mimicking a constitutively protonated D240 (able to coordinate a water molecule) and exhibiting accelerated Na + /Ca 2+ and Ca 2+ /Ca 2+ exchange rates [25]. These data suggest that the deprotonation of D240 is not required for transport catalysis in NCX_Mj, even though transient deprotonation might occur initially during ion recognition.…”

“…The structural information gained from the X-ray studies of NCX_Mj makes this protein an ideal system for studying ion transport by NCX proteins, since NCX_Mj lacks regulatory domains, while exhibiting striking similarities with mammalian NCXs in ion transport machinery [24,25,26,27,35]. The Khananshvili laboratory has extensively studied the functional properties of NCX_Mj by using 45 Ca 2+ -uptake assays in Escherichia coli -derived vesicles containing overexpressed NCX_Mj protein and its mutants (Figure 3A).…”

Computational ‘microscopy’ of cellular membranes

“…State-of-the-art examples are studies of membrane binding and the formation of pores by antimicrobial and cell-penetrating peptides (Berglund et al, 2015;Huang and García, 2013;Moiset et al, 2013;Ulmschneider et al, 2012), of the specific binding of lipids to membrane proteins (Aponte-Santamaria et al, 2012;Contreras et al, 2012;Lee and Lyman, 2012;Pöyry et al, 2013), of the cyclodextrin-mediated extraction of cholesterol from membranes (López et al, 2013a), of the dynamic organization of multi-component membranes (Martinez-Seara et al, 2010;Sodt et al, 2014;Wu et al, 2014b), of lipid-peptide interplay in membrane fusion (Blanchard et al, 2014;Larsson and Kasson, 2013) and of the functioning of membrane proteins (Dror et al, 2011;Kopfer et al, 2014;Maffeo et al, 2012;Marinelli et al, 2014;Moradi et al, 2015;Ostmeyer et al, 2013;Romo et al, 2010).…”