A Common Ca2+-Driven Interdomain Module Governs Eukaryotic NCX Regulation

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

Structure-Based Engineering of Lithium-Transport Capacity in an Archaeal Sodium–Calcium Exchanger

Members of the Ca²⁺/cation exchanger superfamily (Ca²⁺/CA) share structural similarities (including highly conserved ion-coordinating residues) while exhibiting differential selectivity for Ca²⁺, Na⁺, H⁺, K⁺, and Li⁺. The archaeal Na⁺/Ca²⁺ exchanger (NCX_Mj) and its mammalian orthologs are highly selective for Na⁺, whereas the mitochondrial ortholog (NCLX) can transport either Li⁺ or Na⁺ in exchange with Ca²⁺. Here, structure-based replacement of ion-coordinating residues in NCX_Mj resulted in a capacity for transporting either Na⁺ or Li⁺, similar to the case for NCLX. This engineered protein may serve as a model for elucidating the mechanisms underlying ion selectivity and ion-coupled alternating access in NCX and similar proteins

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

International audienceIn analogy with many other proteins, Na(+)/Ca(2+) 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(+)/Ca(2+) 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 Ca(2+) 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

Dynamic distinctions in the Na + /Ca 2+ exchanger adopting the inward- and outward-facing conformational states

International audienceNa(+)/Ca(2+) exchanger (NCX) proteins operate through the alternating access mechanism, where the ion-binding pocket is exposed in succession either to the extracellular or the intracellular face of the membrane. The archaeal NCX_Mj (Methanococcus jannaschii NCX) system was used to resolve the backbone dynamics in the inward-facing (IF) and outward-facing (OF) states by analyzing purified preparations of apo- and ion-bound forms of NCX_Mj-WT and its mutant, NCX_Mj-5L6-8. First, the exposure of extracellular and cytosolic vestibules to the bulk phase was evaluated as the reactivity of single cysteine mutants to a fluorescent probe, verifying that NCX_Mj-WT and NCX_Mj-5L6-8 preferentially adopt the OF and IF states, respectively. Next, hydrogen-deuterium exchange-mass spectrometry (HDX-MS) was employed to analyze the backbone dynamics profiles in proteins, preferentially adopting the OF (WT) and IF (5L6-8) states either in the presence or absence of ions. Characteristic differences in the backbone dynamics were identified between apo NCX_Mj-WT and NCX_Mj-5L6-8, thereby underscoring specific conformational patterns owned by the OF and IF states. Saturating concentrations of Na(+) or Ca(2+) specifically modify HDX patterns, revealing that the ion-bound/occluded states are much more stable (rigid) in the OF than in the IF state. Conformational differences observed in the ion-occluded OF and IF states can account for diversifying the ion-release dynamics and apparent affinity (Km ) at opposite sides of the membrane, where specific structure-dynamic elements can effectively match the rates of bidirectional ion movements at physiological ion concentrations

G503 Is Obligatory for Coupling of Regulatory Domains in NCX Proteins

In multidomain proteins, interdomain linkers allow an efficient transfer of regulatory information, although it is unclear how the information encoded in the linker structure coins dynamic coupling. Allosteric regulation of NCX proteins involves Ca²⁺-driven tethering of regulatory CBD1 and CBD2 (through a salt bridge network) accompanied by alignment of CBDs and Ca²⁺ occlusion at the interface of the two CBDs. Here we investigated “alanine-walk” substitutions in the CBD1–CBD2 linker (501-HAGIFT-506) and found that among all linker residues, only G503 is obligatory for Ca²⁺-induced reorientations of CBDs and slow dissociation of occluded Ca²⁺. Moreover, swapping between positions A502 and G503 in the CBD1–CBD2 linker results in a complete loss of slow dissociation of occluded Ca²⁺, meaning that dynamic coupling of CBDs requires an exact pose of glycine at position 503. Therefore, accumulating data revealed that position 503 occupied by glycine is absolutely required for Ca²⁺-driven tethering of CBDs, which in turn limits the linker’s flexibility and, thus, restricts CBD movements. Because G503 is extremely well conserved in eukaryotic NCX proteins, the information encoded in G503 is essential for dynamic coupling of the two-domain CBD tandem and, thus, for propagation of the allosteric signal

Structure of the CBD12 tandem.

<p>(A) Crystal structure of CBD12-E454K in cartoon representation. CBD1 and CBD2 are colored orange and red, respectively. The rectangles frame a zoom perspective as depicted in panels B (blue), C (magenta) and D (green). Green and blue spheres depict Ca²⁺ ions and water molecules, respectively. Dotted black lines denote electron density chain breaks in the protein. (B–D) Residues with buried surfaces in the interface are depicted as sticks, with their electron density contoured at 1.5 σ (blue mesh).</p