A common Ca2+-driven interdomain module governs eukaryotic NCX regulation

Abstract

Na(+)/Ca(2+) exchanger (NCX) proteins mediate Ca(2+)-fluxes across the cell membrane to maintain Ca(2+) homeostasis in many cell types. Eukaryotic NCX contains Ca(2+)-binding regulatory domains, CBD1 and CBD2. Ca(2+) 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 Ca(2+). 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 Ca(2+) binding to Ca3-Ca4 sites tethers the domains via a network of interdomain salt-bridges. This Ca(2+)-driven interdomain switch controls slow dissociation of "occluded" Ca(2+) from the primary sensor and thus dictates Ca(2+) sensing dynamics. In the Ca(2+)-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 Ca(2+)-driven interdomain switch described here represents a general mechanism for initial conduction of regulatory signals in NCX variants.

Figure 1. Structure of the CBD12 tandem.

(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).

Figure 2. Conservation analysis of CBD domains.

(A) CBD12-E454K structure colored according to the conservation score of each residue. (B) Conservation score for each residue. Negative values indicate conservation while positive values indicate variability. The sequence refers to the cardiac splice-variant, which is 35 residues longer than the brain spliced variant used in our study. (C) Conservation of interface residues located on the G-strand of CBD1 and BC-loop of CBD2.

Figure 3. Ca²⁺ binding sites.

Ca²⁺ coordination in the CBD12-E454K crystal structure (orange) and in the CBD1-WT crystal structure (cyan, PDB 2DPK). Residues coordinating Ca²⁺ are depicted as sticks.

Figure 4. Stopped flow analysis of CBD12-E454K and CBD12-R532A

. (A) Monophasic (uncoupled CBDs) and biphasic (coupled CBDs) dissociation kinetics of two Ca²⁺ ions from the Ca3-Ca4 sites, measured by stopped-flow techniques. Occupied sites are denoted by filled circles, whereas open circles represent empty sites. (B) Representative traces of Ca²⁺ dissociation kinetics from CBD12-WT, CBD12-E454K and CBD12-R532A. Ca²⁺ dissociation kinetics of CBD12-WT were fit to a double exponential curve with k_f = 5.3±0.04 s⁻¹ and k_s = 0.57±0.001 s⁻¹. The trace of CBD12-E454K was fit to a double exponential curve with k_f = 52.2±1.04 s⁻¹ and k_s = 0.73±0.001 s⁻¹. The representative trace of CBD12-R532A was fit to a single exponential curve with k_f = 3.6±0.01 s⁻¹. (C) Bars represent the mean ± S.E values of the “fast” phase (k_f) of Ca²⁺ dissociation (n = 6) and the mean ± S.E of slow off-rates (k_s) (n = 6). For CBD12-R532A, in which monophasic dissociation is observed, k _f and k _s are identical.

Figure 5. SAXS analysis of CBD12 proteins

Bead model reconstruction was performed for each protein on the basis of experimental SAXS measurements. The experiments were done in the absence (left column) and presence (right column) of Ca²⁺, for the wild-type CBD12-WT (A), CBD12-7A (B) CBD12-R532A (C) and CBD12-E454K (D). The CBD1 domain (orange, PDB code: 2FWS) and the CBD2 domain (red, PDB code: 2FWU) are shown as cartoon and surface, and manually fit to the bead model shape (blue mesh), excepting panel A and D, right, where the cartoon is a depiction of the current crystal structure.

Figure 6. Superposition of CBD12 from NCX and CALX.

The structures of CBD12-E454K from NCX1 and of CBD12-1.1 and CBD12-1.2 from CALX (PDB codes 3RB5 and 3RB7, respectively) are colored cyan, black and green, respectively. The indicated values represent the hinge angle between CBD1 and 2 as defined by Cα atoms K373, H501 and E647 (NCX CBD12-E454K) and R443, H553 and I692 (CALX CBD12-1.1 and CBD12-1.2).