Asymmetric Preorganization of Inverted Pair Residues in the Sodium-Calcium Exchanger

Abstract

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.

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

“…Therefore, D240 is protonated in both the Ca 2+ and Na + translocation steps, whereas, in the ground state, both ions do not bind to S mid . This interpretation is further supported by the lack of an effect of the N81A mutation either on the Na + /Ca 2+ or Ca 2+ /Ca 2+ exchange rates (notably, N81 is the only residue that exclusively belongs to the S mid site, whereas the three other residues at S mid , E54, E213, and D240 participate in ion-coordination at other sites as well) [26]. Notably, kinetic analyses suggest that D240 may contribute to the transition state stabilization of ion-bound species, even though neither Ca 2+ nor Na + occupies the S mid site in the ground state [26].…”

“…This interpretation is further supported by the lack of an effect of the N81A mutation either on the Na + /Ca 2+ or Ca 2+ /Ca 2+ exchange rates (notably, N81 is the only residue that exclusively belongs to the S mid site, whereas the three other residues at S mid , E54, E213, and D240 participate in ion-coordination at other sites as well) [26]. Notably, kinetic analyses suggest that D240 may contribute to the transition state stabilization of ion-bound species, even though neither Ca 2+ nor Na + occupies the S mid site in the ground state [26]. The protonation of D240 during the transport cycle also provides a simple explanation for the “confusing” observation that the conserved residue at position 240 in known prokaryotic and eukaryotic Na + /Ca 2+ exchangers is an asparagine, and not an aspartate.…”

“…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].…”

“…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).…”

Conformational dynamics of a neurotransmitter:sodium symporter in a lipid bilayer

“…To achieve these goals, we incorporated LeuT, a thermostable prokaryotic NSS homolog, into lipid bilayer nanodiscs. To circumvent the need to label specific residues, we monitored the conformational hallmarks of LeuT using HDX-MS, a technique that has previously been used to probe the conformational dynamics of other membrane proteins (46)(47)(48). We biased the conformational equilibrium to more outwardfavoring orientations by adding saturating Na + to WT LeuT.…”