Key residues controlling bidirectional ion movements in Na+/Ca2+ exchanger

Abstract

Prokaryotic and eukaryotic Na⁺/Ca²⁺ exchangers (NCX) control Ca²⁺ homeostasis. NCX orthologs exhibit up to 10⁴-fold differences in their turnover rates (k_cat), whereas the ratios between the cytosolic (cyt) and extracellular (ext) K_m values (K_int = K_m^Cyt/K_m^Ext) are highly asymmetric and alike (K_int ≤ 0.1) among NCXs. The structural determinants controlling a huge divergence in k_cat at comparable K_int remain unclear, although 11 (out of 12) ion-coordinating residues are highly conserved among NCXs. The crystal structure of the archaeal NCX (NCX_Mj) was explored for testing the mutational effects of pore-allied and loop residues on k_cat and K_int. Among 55 tested residues, 26 mutations affect either k_cat or K_int, where two major groups can be distinguished. The first group of mutations (14 residues) affect k_cat rather than K_int. The majority of these residues (10 out of 14) are located within the extracellular vestibule near the pore center. The second group of mutations (12 residues) affect K_int rather than k_cat, whereas the majority of residues (9 out 12) are randomly dispersed within the extracellular vestibule. In conjunction with computational modeling-simulations and hydrogen-deuterium exchange mass-spectrometry (HDX-MS), the present mutational analysis highlights structural elements that differentially govern the intrinsic asymmetry and transport rates. The key residues, located at specific segments, can affect the characteristic features of local backbone dynamics and thus, the conformational flexibility of ion-transporting helices contributing to critical conformational transitions. The underlying mechanisms might have a physiological relevance for matching the response modes of NCX variants to cell-specific Ca²⁺ and Na⁺ signaling.

Fig. 1.

Structure of NCX_Mj. (A) Crystal structure of 3Na⁺-bound NCX_Mj (PDB 5HXE) in cartoon representation. The symmetry-related two halves are colored in gray (TM1–5) and blue (TM6–10), respectively. Purple and red spheres represent Na⁺ ions and water molecule, respectively. (B) Superposition of the symmetry-related two halves, as colored in panel A. (C–F) The ion-binding sites of NCX_Mj. The S_mid site is not shown, since there is no experimental or computational evidence that this site can bind either Na⁺ or Ca²⁺. Ion-coordinating residues are shown as sticks. Purple and green spheres represent Na⁺ and Ca²⁺ ions, respectively.

Fig. 2.

Mutational effects on the V_max values of the Na⁺/Ca²⁺ exchange. (A) The initial rates (t = 5s) of Na⁺-dependent ⁴⁵Ca²⁺-uptake were measured by using E. coli-derived vesicles containing the overexpressed protein of a given mutant or WT NCX_Mj (see Materials and Methods). The V_max values of the Na⁺/Ca²⁺ exchange reaction were measured for NCX_Mj mutants as described in Materials and Methods. The V_max values of the indicated mutants are presented in percentage values in comparison with WT V_max (100%). Data are presented as bars (mean ± SE). The data were derived from at least 3 independent experiments. Residues are colored according to their mutational effects on V_max, as indicated. (B) Topological positions of mutated residues are presented according to color assignments, described in panel A. (C) Cartoon representation of NCX_Mj. Mutated residues are shown as spheres and are displayed in color according to the mutational effect (see panel A). Note the specific distribution of residues belonging to group 1 (red) in the vicinity of the pore core.

Fig. 3.

Mutational effects on the K_m^Cyt values of the Na⁺/Ca²⁺ exchange. (A) The K_m^Cyt values of the Na⁺/Ca²⁺ exchange reaction were measured for the indicated mutants, as described in Materials and Methods. The K_m^Cyt values of the indicated mutants are presented in percentage values in comparison with the WT K_m^cyt values. Data were obtained from at least 3 independent experiments and are presented as mean ± SE. Residues are colored according to their effects on K_m, as indicated. (B) Topological positions of mutated residues are presented in color, as displayed in the panel A. (C) Cartoon presentation of NCX_Mj. Mutated residues are shown as spheres and colored according to the mutational effects, as in panel A.

Fig. 4.

Mutational effects of pore-forming residues on the K_int and k_cat values of the Ca²⁺/Ca²⁺ exchange. (A) Schematic representation of the Ca²⁺/Ca²⁺ exchange reaction for measuring the K_int and k_cat values of bidirectional Ca²⁺ movements. The dashed line represents the partial reaction of the Ca²⁺/Ca²⁺ exchange, where the intrinsic equilibrium of bidirectional Ca²⁺ movements (K_int) is defined as the ratio of apparent affinities for Ca²⁺ at the cytosolic (K_m^Cyt) and extracellular (K_m^Ext) sides (K_int = K_m^Cyt/K_m^Ext). B) The initial rates (t = 5s) of Ca²⁺-dependent ⁴⁵Ca²⁺-uptake were measured for determining the K_m^Cyt and K_m^Ext values. The K_m^Cyt values were determined using varying [Ca²⁺]_Cyt = 2–200 μM and saturating [Ca²⁺]_Ext = 2 mM (blue bars), whereas the K_m^Ext values were measured using varying [Ca²⁺]_Ext = 10–2000 μM and fixed [Ca²⁺]_cyt = 2mM (red bars). The k_cat values were derived from the measured V_max values of the Ca²⁺/Ca²⁺ exchange as described in Materials and Methods. Data are presented as mean ± SE of at least 3 independent experiments. Mutations resulting in K_int <1 or K_int >1 are in green and blue, respectively. (C) The K_m^Cyt (blue bars) and K_m^Ext (red bars) values of the Ca⁺/Ca²⁺ exchange reaction were measured as described in panel B. Data are presented as mean ± SE obtained at least 3 independent experiments (see Materials and Methods). Residues are colored according to their mutational effects on K_int, as shown in panel B. (D) Topological positions of residues are assigned according to their mutational effects on K_int, as colored in panel B. (E) Cartoon presentation of NCX_Mj. Mutated residues are shown as spheres, where they are colored according to mutational effects, described in panel B.

Fig. 5.

Mutational effects of loop-residues on the K_int and k_cat values of the Ca²⁺/Ca²⁺ exchange. (A) Mutational effects on the K_int and k_cat values of the Ca²⁺/Ca²⁺ exchange were analyzed as described in Fig. 4 (see also Materials and Methods). Data were derived from at least 3 independent experiments and are presented as mean ± SE. Mutations exhibiting K_int < < 1, K_int ≈ 1 and K_int > > 1 are in green, blue, and red, respectively. (B) Cartoon presentation of NCX_Mj. Mutated residues are shown as spheres, where the colored residues are assorted according to their mutational effects, as indicated in panel A. (C) The measured values of K_m^Cyt (blue bars) and K_m^Ext (red bars) of the Ca⁺/Ca²⁺ exchange reaction were derived from at least 3 independent experiments. Data are presented as mean ± SE. Residues are colored according to their mutational effects on K_int, as colored in panel A.

Fig. 6.

Spatial distribution of residues differentially affecting the V_max and K_int values. (A) For each mutant, the V_max value of the Ca²⁺/Ca²⁺ exchange was plotted vs. its position within the pore. The residue-positions are expressed as the distance from the pore center (the “zero” represents the position of the pore center). The residues significantly affecting the V_max value (< 20% of WT V_max) are shown in blue, where less significant residues affecting the ion-transport rates are shown as solid black circles. The solid and dashed blue circles represent the ion-coordinating and non-coordinating residues, respectively. (B) Topological presentation of mutated positions. The mutational effects of residues on V_max are colored as in panel A. (C) For each mutant, the K_int value was plotted vs its distance from the pore center. The mutants exhibiting high K_int values are indicated in blue, whereas the solid and dashed circles represent the pore-forming and helix-loop residues, respectively. Solid black cycles represent mutations having an insignificant effect on K_it (D). Topological presentation of mutated positions are colored as in panel C.

Fig. 7.

Structural-dynamics and functional relationships in NCX_Mj. Schematic heat maps of the hydrogen-deuterium exchange were overlaid on the pore-forming TMs by using the available HDX-MS data [31] and the crystal structural of NCX_Mj [12,30]. The color ruler represents the HDX levels (in %), thereby depicting the characteristic profiles of backbone dynamics in apo NCX_Mj (blue signifies the most rigid and water inaccessible segments in protein). (A) The key residues limiting the ion transport rates (shown as spheres) are located on the TM2B, TM3 A, TM7BC, and TM8 A segments within the pore core, while showing striking differences in the local backbone dynamics at respective locations. (B) Key residues affecting the intrinsic equilibrium (K_int) of bidirectional Ca²⁺ movements (shown as spheres) are distributed at peripheral locations on TM2 A, TM3 A, TM7AB, and TM8 AB, which is consistent with the known mechanisms of ion occlusion and also provides new clues for the mechanisms underlying ion-coupled alternating access.

Fig. 8.

Conformational dynamics of NCX_Mj predicted by elastic network models. (A) Comparison of the theoretically predicted root-mean-square fluctuations (RMSFs) of residues (green) with the B-factors profile (black) observed in X-ray crystallographic experiments (PDB: 3V5S). (B) ANM mode 2 (green arrows) in the presence of lipid bilayer induce the closure of the extracellular vestibule, in favor of a transition from OF open state to an OF occluded state. (C) Cross-correlation map for the coupled fluctuations of all residues driven by the softest GNM modes 1–3. Red regions indicate pairs of residues undergoing correlated movements, blue regions indicate the pairs that undergo anticorrelated (coupled but opposite direction) fluctuations. Note that TM1 and TM6 are highly correlated, and they are anticorrelated with TM2–4 and TM8–9. (D) Residues in group 1 occupy minima in the mobility profile. The graph displays global mobilities of residues (corresponding to the soft modes 1, 2 and 3) obtained by GNM analysis of NCX_Mj embedded in a lipid bilayer. The blue, red and oranges curves represent the predictions from the respective three GNM modes. Red, blue and green spheres indicate the mutated residues belonging to those classified as Group 1 (V_max < 20%), Group 2 (30 < V_max < 70%), and Group 3 (> 80%), respectively in Fig. 2. The residues that show minimal (close to zero) mobility in at least one of the three soft modes are shown by their color-coded sphere. Those located in other regions, are indicated on the mode 2 curve. Minima refer to structural regions that serve as hinges or anchors in the global movements of the entire exchanger. All calculations and visualizations were performed using the DynOmics server.

Fig. 9.

Conformational dynamics of NCX_Mj captured by full-atomic computations suggests the potential role of the 5L6 loop. (A–B) Simulations indicate that the 5L6 loop reorients differently in the outward-facing (OF) and inward-facing (IF) conformers of NCX_Mj as illustrated for (A) X-ray resolved NCX_Mj OF conformer (PDB: 5HWY), and (B) MD-refined IF conformer. (C) Structure of the IF homologous superfamily member VCX1 Calcium/Proton Exchanger resolved in the IF state (PDB: 4K1C). The simulations suggest that the 5L6 loop (orange) swings into the intracellular vestibule in the IF state. Notably, the homologous 5L6 loop (or acidic helix) of VCX1 (H⁺/Ca²⁺ exchanger) also exhibits a similar orientation in the IF state.