Crystal Structures of Progressive Ca²⁺ Binding States of the Ca²⁺ Sensor Ca²⁺ Binding Domain 1 (CBD1) from the CALX Na⁺/Ca²⁺ Exchanger Reveal Incremental Conformational Transitions

Abstract

Na⁺/Ca²⁺ exchangers (NCX) constitute a major Ca²⁺ export system that facilitates the re-establishment of cytosolic Ca²⁺ levels in many tissues. Ca²⁺ interactions at its Ca²⁺ binding domains (CBD1 and CBD2) are essential for the allosteric regulation of Na⁺/Ca²⁺ exchange activity. The structure of the Ca²⁺-bound form of CBD1, the primary Ca²⁺ sensor from canine NCX1, but not the Ca²⁺-free form, has been reported, although the molecular mechanism of Ca²⁺ regulation remains unclear. Here, we report crystal structures for three distinct Ca²⁺ binding states of CBD1 from CALX, a Na⁺/Ca²⁺ exchanger found in Drosophila sensory neurons. The fully Ca²⁺-bound CALX-CBD1 structure shows that four Ca²⁺ atoms bind at identical Ca²⁺ binding sites as those found in NCX1 and that the partial Ca²⁺ occupancy and apoform structures exhibit progressive conformational transitions, indicating incremental regulation of CALX exchange by successive Ca²⁺ binding at CBD1. The structures also predict that the primary Ca²⁺ pair plays the main role in triggering functional conformational changes. Confirming this prediction, mutagenesis of Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, produces dramatic reductions of the regulatory Ca²⁺ affinity for exchange current, whereas mutagenesis of Glu⁵²⁰, which coordinates the secondary Ca²⁺ pair, has much smaller effects. Furthermore, our structures indicate that Ca²⁺ binding only enhances the stability of the Ca²⁺ binding site of CBD1 near the hinge region while the overall structure of CBD1 remains largely unaffected, implying that the Ca²⁺ regulatory function of CBD1, and possibly that for the entire NCX family, is mediated through domain interactions between CBD1 and the adjacent CBD2 at this hinge.

Introduction

The Na⁺/Ca²⁺ exchanger (NCX)³ plays an important role in eukaryotic Ca²⁺ homeostasis. This transporter functions as a Ca²⁺ efflux mechanism across cell membranes and broadly participates in Ca²⁺-mediated cellular signaling. NCXs have been identified in numerous tissues and cell types from several different species. In cardiac muscle, NCX1.1 plays a critical role in transsarcolemmal Ca²⁺ efflux, an essential requirement for cardiac relaxation (1). In neuronal tissues, a variety of exchangers are intricately involved in the control of excitation-secretion signaling (2). Notably, all characterized mammalian Na⁺/Ca²⁺ exchangers exhibit a common Ca²⁺-dependent regulatory mechanism, whereby their activity requires the presence of low concentrations of Ca²⁺ on their intracellular surface, and their activity is augmented in parallel with elevated intracellular Ca²⁺ levels (3). This important regulatory property may permit the timely coupling of exchange function to alterations in intracellular Ca²⁺ concentrations to meet the continuous needs for overall Ca²⁺ balance.

The general similarities of exchange function and regulatory properties within the large NCX protein family are ascribed to their conserved structural arrangements: nine predicted transmembrane (TM) segments form the ion translocation pathway and a large loop of ∼500 amino acid residues splits TM helix-5 and -6 on the intracellular side of the molecule (4). Ca²⁺-dependent regulation is attributed exclusively to Ca²⁺ interactions on the intracellular loop (5). A pair of Ca²⁺ binding domains (CBD1 and -2), called CALX-β motifs, has been identified (6). Sequence analysis revealed that CBD1 has conserved Ca²⁺ binding sites throughout the NCX family, whereas greater sequence diversity and/or Ca²⁺ binding capabilities occurs in CBD2 (7, 27). Given that CBD1 exhibits a higher Ca²⁺ affinity than CBD2 (8), it has been suggested that CBD1 acts as the primary sensor in the pair of CBDs. Mutations of carboxylate residues at CBD1 result in a pronounced reduction of the affinity for functional Ca²⁺ regulation (9). The Ca²⁺-bound structures of CBD1 of NCX1 have recently been determined by NMR, and more recently by x-ray crystallography (8, 11). The crystal structures revealed that four Ca²⁺ ions bind at the end of a β-sandwich structure of CBD1. More recently, a detailed study by backbone NMR suggested that Ca²⁺ binding induces a selective conformational change of CBD1 limited to the residues in the binding site, whereas the core of the β-sandwich structure remains unaffected (12). These observations raise a fundamental question of how the primary sensor role of CBD1 is conducted to the TM segments to control exchange activity.

There is currently no structure available for the apoform of CBD1. Consequently, there is no related mechanistic information or insight into how Ca²⁺ binding induces the conformational change of CBD1 required for transduction of this signal. In the reported Ca²⁺-bound NCX1-CBD1 crystal structure, the Ca²⁺ binding site was saturated with four Ca²⁺ atoms (11). Whether these four Ca²⁺ access the binding site of CBD1 simultaneously or in a sequential way is unknown. Information of this type is critical toward understanding whether exchange function is simply switched on or off by Ca²⁺ or whether various degrees of exchange function are graded by different levels of Ca²⁺ occupancy.

CALX, a Na⁺/Ca²⁺ exchange protein, was first identified in Drosophila photoreceptor cells (6, 13, 24). CALX is responsible for extruding intracellular Ca²⁺ from these cells and plays an essential role in light-mediated signaling in Drosophila sensory neurons (14). CALX shares 49% amino acid identity with the prototypical canine Na⁺/Ca²⁺ exchanger, NCX1.1. Functionally, it shares many properties found in mammalian exchangers (13). However, CALX exhibits a completely opposite response to regulatory Ca²⁺ compared with all other characterized mammalian NCX homologs: the highest activity of CALX occurs in the complete absence of regulatory Ca²⁺ and its activity is progressively inhibited, rather than stimulated, by elevations in intracellular regulatory [Ca²⁺]. This negative Ca²⁺ regulation of CALX leads to a considerable loss of signal amplification in the light response of the Drosophila visual system that triggers the photoreceptor cell cascade (14). CALX also possesses a pair of CBD domains on its intracellular loop. The mechanism underlying the negative Ca²⁺ regulatory phenotype observed for CALX is still elusive and enigmatic based on the existing structural information for NCX1. Our recent crystal structure of CALX-CBD2 showed that this site is not a functional Ca²⁺ binding site, suggesting that CBD1 must be the critical site involved in Ca²⁺ regulation of CALX (7). Furthermore, structural predictions, together with previous mutagenesis studies (7, 15) have strongly suggested that CALX-CBD1 possesses a similar Ca²⁺ binding site as does NCX1. To gain further insight into the Ca²⁺-binding mechanisms of Na⁺/Ca²⁺ exchange proteins in atomic detail and to investigate the negative Ca²⁺ regulatory property of CALX, we have determined crystal structures of CBD1 from CALX1.1 in the presence and absence of Ca²⁺ and studied the properties of this regulatory mechanism by mutagenesis and electrophysiology.

EXPERIMENTAL PROCEDURES

Expression and Purification of CALX-CBD1 Domains

The gene fragments encoding the CBD1 (amino acids 442–554) from a full length cDNA of Drosophila CALX1.1 were cloned into the vector pET28a (Novagen) with restriction sites of NdeI/XhoI. In the generated plasmid, CBD1 has an N-terminal His-tag spaced by a thrombin cleavage site. Protein expression was performed in Escherichia coli BL21(DE3) cells in the autoinduction medium (16) overnight at 25 °C. The cell pellet was suspended in a lysis buffer containing 50 mm sodium phosphate, 500 mm NaCl, 20 mm imidazole, pH 8.0, and ruptured by a high pressure homogenizer (Avestin). The lysate supernatant was applied to a nickel-nitrilotriacetic acid resin column (GE Healthcare), and the CALX-CBD1 protein was eluted with 300 mm imidazole. The purified protein was dialyzed overnight against a Tris-buffered saline buffer, pH 7.4, and was incubated with thrombin protease (GE Healthcare) overnight at 4 °C to truncate the His-tag. The proteolytic reaction mixtures were reapplied to nickel-nitrilotriacetic acid resin and the pass-through containing untagged CALX-CBD1 proteins was concentrated by Centricon (Millipore) and further purified by size-exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare). The protein concentration was determined with a Coomassie protein determination kit (Pierce).

Crystallization of CALX-CBD1

All crystallization experiments were performed using the sitting-drop vapor diffusion method at 18 °C. CALX-CBD1, premixed with 1 mm CaCl₂ at a protein concentration of 10 mg/ml, was crystallized using the following conditions: 50 mm MES, pH 6.0, 20% polyethylene glycol 3350. To obtain the apoform crystals, CALX-CBD1 protein was incubated with 10 mm EDTA for 1 h and then dialyzed against Tris-buffered saline buffer, pH 7.4, prior to the crystallization experiment. The apoform crystals were obtained under different conditions using 100 mm Bis-Tris, pH 6.5, 200 mm NH₄Ac, 10 mm MgCl₂, 15% polyethylene glycol 10,000.

Data Collection

All crystals were flash-cooled to 100 K with 25% glycerol as the cryoprotectant. Diffraction data for the Ca²⁺ form crystal of CBD1 were collected at beamline X06SA of the Swiss Light Source (Villigen). A long wavelength (1.90 Å) was used for data collection with the intention of exploiting weak anomalous signals from Ca²⁺ atoms. The data collection for the apoform of CBD1 was carried out at the Advanced Light Sources beam line 4.2.2 (Berkeley, CA).

Data Processing and Structural Determination

Data processing, merging, and reduction were carried out with programs XDS and XSCALE (17). The CALX-CBD1 structures were solved using the molecular replacement method by the program PHASER (18) and using the CBD1 structure from NCX1 (Protein Data Bank code 2DPK) as a search model. Both structures were refined using the program Refmac (19). The model building was performed using COOT (20). Crystallographic data and the model refinement statistics are given in Table 1. The anomalous Fourier map in Fig. 1A was calculated using the program FFT (21) with phases from the final refined coordinates and observed anomalous difference in diffraction data. All figures were prepared using the program PyMOL (22).

TABLE 1.

Data processing and model refinement statistics for CALX-CBD1 structures

	CALX-CBD1 Ca2+-bound form	CALX-CBD1 apoform
Data collection
Space group	P212121	P212121
Unit cell (Å)	59.4, 73.7, 129.7	59.1, 76.5, 129.0
Wavelength (Å)	1.90	1.00
Resolution range (Å)a	20–2.25 (2.3–2.25)	47.6–1.6 (1.70–1.60)
Observations (total/unique)	196,637/26,203	553,318/146,906
I/σ (I)a	14.1 (3.1)	15.6 (2.0)
Completeness (%)a	94.4 (71.8)	98.7 (95.8)
Rsym (%)a	7.2 (54.4)	10.7 (57.7)
Structure refinement
Rwork/Rfree	20.8/26.5	19.2/22.1
Number of atomsb	3,596/16/120	3,482/6/461
r.m.s.d.c bond length (Å)	0.017	0.012
r.m.s.d. bond angle	1.76°	1.63°
Ramachandran analysis (%)
Most favored	91.2	91.5
Additional favored	8.8	8.2
Generously allowed	0	0.3
B factors (average)
Monomer A/B/C/D	47.7/45.3/46.1/62.3	20.4/18.7/21.2/23.2
Ca2+	64.5	25.7
Water	39.1	32.1

^a Values within parentheses refer to the high resolution shell.

^b Non-hydrogen protein atoms/metal atoms/water molecules.

^c r.m.s.d., root mean square deviation.

FIGURE 1.

Crystal structures of CALX-CBD1. A, structural alignment of the CBD1 from CALX and NCX1. CALX-CBD1 shown as rainbow ribbons was superimposed on the NCX1-CBD1 crystal structure shown in gray. The anomalous difference map is displayed in slate and contoured at 6.0σ to indicate the Ca²⁺ positions. The short F–G loop of CALX is highlighted, whereas that from NCX1 is invisible. B, structural alignment of the Ca²⁺ binding sites of CALX-CBD1 and NCX1-CBD1. The Ca²⁺-bound CALX-CBD1 structure in cyan is superimposed onto the NCX1-CBD1 structure (colored in wheat). Four Ca²⁺ and water molecules are represented as green and red spheres, respectively. The residues coordinating Ca²⁺ are shown as stick balls; Ca²⁺ interactions with residues are shown as yellow dashed lines. The label for the E385 residue in NCX1 is shown in underlined italics.

Circular Dichroism (CD) Spectroscopy

Prior to CD spectroscopic analysis, CALX-CBD1 protein used for crystallization was passed through a desalting column (GE Healthcare) equilibrated with a solution containing 200 mm NaF, pH 7.4, to remove any Cl⁻. The protein concentration was adjusted to 1 mg/ml before measurement. CD spectra of the CBD1 protein were collected at room temperature over a wavelength range from 190 to 260 nm with a Jasco J-720 spectrometer using a 0.02-cm cylindrical cell.

Mutational Analysis Using Giant Excised Patch Clamping

Mutations of CALX1.1 were introduced by a modified site-directed mutagenesis procedure (23). To exclude the possibility of random PCR errors on large cNDA of CALX (>3 kb), a minimal DNA fragment containing the sequencing-confirmed mutations was recloned back to the parent vector with appropriate restriction sites.

The effect of each mutation on Ca²⁺ regulation of CALX1.1 was measured by outward Na⁺-Ca²⁺ exchange current recordings using the giant, excised patch clamp technique, as described previously (24). Briefly, Xenopus laevis oocytes were injected with ∼23–35 ng of cRNA of wild-type or mutant CALX1.1 and maintained at 18 °C. Electrophysiological measurements were typically obtained from day 3 to 7 postinjection. Borosilicate glass pipettes were pulled and polished to a final, inner diameter of ∼20–30 μm, and coated with a Parafilm®:mineral oil mixture to enhance patch stability and reduce electrical noise. Oocytes were briefly (∼5–10 min) transferred to a solution containing: 100 mm KOH, 100 mm K-aspartate, 100 mm MES, 20 mm HEPES, 10 mm NH₃SO₃, 5.0 mm EGTA, 5.0 mm Mg(OH)₂; pH 7.0, at room temperature (with MES) to allow sufficient shrinkage of the cells in order for their vitellin layers to be removed by dissection. Giga ohm seals were formed by gentle suction, and membrane patches were excised by progressive movements of the pipette tip. Rapid solution changes were introduced using computer-controlled, multi-channel perfusion devices. Axon Instruments® hardware and software were used for data acquisition. A holding potential of 0 mV was employed for all current measurements.

Pipette solutions contained 100 mm N-methyl-d-glucamine, 100 mm MES, 30 mm HEPES, 30 mm TEA-OH, 16 mm sulfamic acid, 8.0 mm CaCO₃, 6.0 mm KOH, 0.25 mm ouabain, 0.1 mm flufenamic acid, 0.1 mm niflumic acid; pH 7.0, at room temperature (with MES). Currents were elicited by switching from Li⁺- to Na⁺-based perfusion solutions containing: 100 mm NaOH or LiOH, 100 mm l-aspartic acid, 20 mm CsOH, 20 mm HEPES, 20 mm TEA-OH, 10 mm EGTA, 3.00–21.84 mm NH₃SO₃, 0–9.91 mm CaCO₃, 1.00–1.50 mm Mg(OH)₂; pH 7.0, at 30 °C (with MES or LiOH). Stock solutions of CaCO₃ and Mg(OH)₂ were prepared in a ratio of 1:2 with sulfamic acid. Total Mg²⁺ and Ca²⁺ were adjusted to yield free concentrations of 1.00 mm and 0–30 μm, respectively, using MAXC software (25).

All experiments were conducted at 30 °C. Origin® software was used for curve-fitting and statistical analyses. Pooled data are mean ± S.E. Student's t test or one-way analysis of variance and Tukey's post hoc test, were used for statistical determinations. p < 0.05 was considered significant.

RESULTS

Overview of CALX1.1-CBD1 Structure

The Ca²⁺-bound structure of CALX-CBD1 was determined at 2.25 Å resolution. The structure shows CALX-CBD1 has an immunoglobulin-like conformation formed by two anti-parallel β-sheets consisting of β-strands, A, B, G and C, D, E, F, respectively (Fig. 1A). CALX-CBD1 shares 60% sequence identity with that of canine NCX1-CBD1. Consequently, these two structures can be superimposed with a root mean square deviation of 0.84 Å for 113 aligned Cα atoms. The most significant difference between these two CBD1 structures occurs within their F-G loops. CALX-CBD1 has a rather short F–G loop with only 9 residues, whereas its counterpart from NCX1 consists of 28 residues and displays high flexibility, as indicated in the NMR structure (8).

Ca²⁺ Binding Site of CALX-CBD1

The Ca²⁺- bound CALX-CBD1 crystal diffraction data set was collected at 1.9 Å wavelength, allowing the assignment of the bound Ca²⁺ by examination of the anomalous signal (Fig. 1A). Four Ca²⁺ are clustered in the distal loops of the β-sandwich with ∼4 Å equal distance spacing between them. Nine carboxylate residues are involved in the coordination of Ca²⁺ (Fig. 1B). The side chains of these acidic residues are arranged in a zipper-like orientation, forming an extensive carboxylate cluster at the top of this β-sandwich. The E–F loop is the major component in the Ca²⁺ binding site with five of its residues involved in Ca²⁺ coordination. That is, the E–F loop crosses over the Ca²⁺ zipper line clamping Ca-1, -2, and -3 ions through coordination with carboxylate groups of Asp⁵¹⁵, Asp⁵¹⁶, and Glu⁵²⁰ together with the carbonyl oxygen atoms of Asp⁵¹⁷ and Val⁵¹⁸. Compared with Ca-1 or -2, which is penta- or hexacoordinated, Ca-4 is only tricoordinated with Asp⁴⁹⁰, Glu⁵²³, and Glu⁵²⁰. Consequently, Ca-4 displays the highest thermal B factor, suggesting it may be the most mobile species during these ligand interactions.

The Ca²⁺ binding sites of CALX-CBD1 are almost identical to those of NCX1 except for Glu⁴⁵⁵, which locates centrally on the basement of the binding site. Compared with the NCX1 structure, this residue rotates its carboxyl group by 90°, resulting in simultaneous coordination with three Ca²⁺ (Ca-1, Ca-2, and Ca-3) instead of one (Ca-2) shown in the NCX1-CBD1 structure (11). In addition, it appears that the disulfide bridge locking the A–B loop near Glu³⁸⁵ in the NCX1-CBD1 structure is absent in CALX-CBD1, as a valine replaces cysteine at position 453.

Conformational Change in the Apoform Structure

To gain additional structural information regarding Ca²⁺ binding to CBD1, the CBD1 protein was treated with 10 mm EDTA and dialyzed prior to crystallization; the apoform structure was determined at 1.6 Å resolution. The EDTA-treated sample gives the same crystal packing as that of the Ca²⁺-bound form; four monomers assemble in anti-parallel in an asymmetric unit. The overall structure of the apoform CBD1 shows no overall conformational changes compared with the Ca²⁺-bound form except within the Ca²⁺ binding sites. In the Ca²⁺-bound structure, Ca²⁺ binding sites of four monomers in an asymmetric unit were identically and fully occupied. In contrast, the same binding sites exhibit three distinct Ca²⁺ binding states in the apoform structure. Monomer B shows a full occupancy state as shown in the Ca²⁺-form structure, where four Ca²⁺ were found in the Ca²⁺ binding site and all residues involved in Ca²⁺ coordination are clearly visible (Fig. 2A).

FIGURE 2.

Stereo views of conformational changes in the Ca²⁺ binding sites of CALX-CBD1. 2F_o − F_c electron density maps contoured at 1.5σ level are shown in blue. Ca²⁺ and water molecules are represented as green and red spheres, respectively. The residues coordinating Ca²⁺ are shown as stick balls. Hydrogen bond interactions are shown as yellow dashed lines. A, B, and C represent three different conformations from the same asymmetric unit of the apoform CALX-CBD1 structure. A, monomer B (four Ca²⁺-bound); B, monomer A (two Ca²⁺-bound); and C, monomer C (no Ca²⁺-bound).

However, monomer A displays a partial Ca²⁺ binding state. Only Ca-1 and -2 (the primary Ca²⁺ pair) were found at similar positions as in monomer B (Fig. 2B). The absence of Ca-3 and Ca-4 (named as the secondary Ca²⁺ pair) does not result in any significant structural change compared with monomer A with root mean square deviation values of 0.28 Å for backbone atoms and 0.68 Å for all atoms. The conformational change occurs exclusively at Glu⁵²⁰, whose side chain is invisible in the electron density map. The backbone of the entire E–F loop is still clearly resolved and appears to be stabilized by the primary Ca²⁺ pair. However, the thermal B factors of the residues on the E–F loop are considerably increased compared with those in monomer B (Fig. 3). A water molecule at position 265 was found at Ca-3 position, which forms two hydrogen bonds with Glu⁴⁵⁵ and Asp⁴⁹⁰. No density can be observed in the Ca-4 position. The conformations of Asp⁴⁹⁰ and Glu⁵²³ remain unchanged; they are stabilized by a hydrogen bond network with a water molecule at position 324.

FIGURE 3.

Thermal B values versus residues in each monomer of an asymmetric unit of CALX-CBD1 structure. A, monomer B; B, monomer A; C, monomer C; and D, monomer D.

Strikingly, both monomers C and D represent the true apoform, and no Ca²⁺ can be found in corresponding regions (Fig. 2C). Nearly all residues involved in Ca²⁺ binding become invisible, particularly the entire E–F loop (Asp⁵¹⁶–Glu⁵²²), which strongly suggests that the primary Ca²⁺ pair plays a critical role in stabilizing the entire Ca²⁺ binding region. The disorder of the Ca²⁺ binding region has no impact on the overall β-sheet structure of CBD1 and the neighboring C–D loop containing Asp⁴⁹⁰ is still clearly resolved. Both Ca²⁺-free forms superimpose well with similar root mean square deviation values of 0.44 Å for backbone atoms and 0.97 Å for all atoms. Notably, no Ca²⁺ binding region from these four monomers is involved in crystal packing, and all four monomers display comparable thermal B factors (Table 1 and Fig. 3).

Our structural observations indicate that no gross conformational changes occur during Ca²⁺ binding. To exclude any possible effects of crystallization constraints, the Ca²⁺ extraction experiment was performed in solution with monitoring by CD spectroscopy. The result shows CBD1 protein exhibits a full β-strand conformation, as expected (Fig. 4A). No detectable change of protein secondary structure was observed by addition of either 2 mm Ca²⁺ (Fig. 4B) or 10 mm EDTA (Fig. 4C). Notably, 10 mm EDTA causes Ca²⁺ unbinding of CBD1 of NCX1 (8).

FIGURE 4.

CD spectra of CALX-CBD1 upon Ca²⁺ binding. A, CBD1 protein after the protein purification; B, the CBD1 protein with 2 mm Ca²⁺; and C, the CBD1 protein with 10 mm EDTA.

Mutational Analysis of Ca²⁺ Binding Site of CBD1

Two residues, Glu⁴⁵⁵ and Glu⁵²⁰, are involved in the coordination of three Ca²⁺ at the CBD1 site, although they differ in the specific Ca²⁺ ions which are involved (Glu⁴⁵⁵ with Ca-1, -2, and -3; Glu⁵²⁰ with Ca-2, -3, and -4). Our structural data suggest that Glu⁴⁵⁵, which coordinates the primary Ca²⁺ pair, plays a more important role in stabilizing the entire Ca²⁺ binding region than Glu⁵²⁰. To examine whether these two glutamate residues have unequal functions in the Ca²⁺-dependent regulatory mechanism, they were mutated into Asp and/or Ala. RNA from either the wild-type CALX1.1 or various mutant exchangers was injected into Xenopus laevis oocytes, and the outward currents were recorded to evaluate functional Ca²⁺ regulation. The overlapping traces in Fig. 5A show representative outward Na⁺-Ca²⁺ exchange currents for wild-type CALX1.1 at four different Ca²⁺ concentrations. In the absence of regulatory Ca²⁺, CALX is fully activated by the application of 100 mm Na⁺. However, both peak and steady-state currents become substantially suppressed by increasing the concentration of regulatory Ca²⁺. (Results obtained at 1, 3, and 10 μm are shown.) The corresponding IC₅₀s for peak and steady-state currents are 0.4 ± 0.1 μm and 0.13 ± 0.002 μm, respectively.

FIGURE 5.

Representative outward Na⁺/Ca²⁺ exchange currents showing the regulatory Ca²⁺ dependence of the wild-type and various CALX1.1 mutant exchangers. 8 mm Ca²⁺ was present in the pipette, and the currents were activated by the addition of 100 mm Na⁺ to the cytoplasmic surface of the patch with regulatory Ca²⁺ absent or present at various concentrations. For wild-type (A), E455D (C) and E455A (D), exchange currents were recorded at four different Ca²⁺ concentrations (0, 1, 3, and 10 μm) as indicated. For E520A (C), only three different Ca²⁺ concentrations (0, 1, and 3 μm) were tested. Ca²⁺_i dependence of pure outward currents for peak (E) and steady state (F) mediated by wild-type CALX1.1 and various mutants. Currents were normalized to those obtained at 0 Ca²⁺_i within the same patch. Data points were averaged from four to seven individual patches. IC₅₀ for peaks: 0.4 ± 0.1, 1.2 ± 0.01, 6.7 ± 1.3, and 8.1 ± 0.5 μm Ca²⁺ for CALX1.1, E520A, E455D, and E455A, respectively. IC₅₀ for steady-state currents: 0.13 ± 0.002, 0.22 ± 0.06, 1.7 ± 0.1, and 3.5 ± 0.2 μm Ca²⁺ for CALX1.1, E520A, E455D, and E455A, respectively.

Even though Glu⁵²⁰ appears to have the equivalent Ca²⁺ coordinating capacity as Glu⁴⁵⁵ (i.e. three Ca²⁺), it exhibits far less importance in mediating the Ca²⁺ regulatory response. Mutations of these two residues illustrate their different functional roles. The Ca²⁺ dependence of peak and steady-state currents of these mutants are presented in Fig. 5, E and F. In the E520A mutation, there is only a slight reduction in the inhibitory potency of regulatory Ca²⁺ for peak currents, (IC₅₀s = 1.2 ± 0.01 μm) and even less for steady-state currents (0.22 ± 0.06 μm) (Fig. 5B).

In sharp contrast, the Ca²⁺ response is much more sensitive to changes at residue Glu⁴⁵⁵. The E455A mutation results in a dramatic alteration of the Ca²⁺ regulatory response. Both peak and steady-state currents are not appreciably inhibited until the level of regulatory Ca²⁺ reaches 3 μm (Fig. 5D). The apparent Ca²⁺ affinity of E455A for regulation of peak currents is reduced by ∼20-fold to IC₅₀ = 8.1 ± 0.5 μm. Steady-state currents show an even greater reduction in the inhibitory potency of regulatory Ca²⁺, which drops by 35-fold to 3.5 ± 0.2 μm. We also mutated Glu⁴⁵⁵ to an Asp. Similar to E455A, as shown in Fig. 5C, this conservative mutant shows a large shift in the affinity for functional Ca²⁺ regulation, which now appears at the level of 1 μm Ca²⁺. The inhibitory potency of regulatory Ca²⁺ is reduced by nearly ∼17-fold for both peak (6.7 ± 1.3 μm) and steady-state currents (1.7 ± 0.1 μm). These results clearly demonstrate that residue Glu⁴⁵⁵ is essential for Ca²⁺ binding and ultimately the transduction of the regulatory Ca²⁺ binding signal. The integrity of Glu⁴⁵⁵ is critical in maintaining normal exchanger regulation of CALX.

DISCUSSION

Ca²⁺ interactions occurring at CBD1 are essential for properly controlling sodium-calcium exchange activity and for the maintenance and reestablishment of resting Ca²⁺ levels in living cells. In this study, the Ca²⁺-bound structure of CALX-CBD1 precisely confirms the occupancy of four Ca²⁺ within this site by their anomalous signals. Our data shows that CALX has a similar overall structure and Ca²⁺ binding site of CBD1 as does the mammalian NCX1, irrespective of their opposite Ca²⁺ regulatory phenotypes. Therefore, Ca²⁺ binding within the CBD1 structures likely represents a general mechanism within the larger NCX family.

Despite the nearly identical sequence composition of the Ca²⁺ binding sites within CBD1 from either CALX or NCX1, both the Ca²⁺-bound and apoform CALX-CBD1 structures consistently demonstrate that Glu⁴⁵⁵ in the core of the Ca²⁺ binding site plays a significantly different role in Ca²⁺ coordination compared with Glu³⁸⁵ in the NCX1 structure. Possibly, this difference between CALX and NCX1 could be attributed to the lower resolution (2.5 Å) of the NCX1 structure (11). The oxygen atoms of the carboxylate group of Glu³⁸⁵ were not well defined, as seen in the electron density of the NCX1 structure. To date, no mutations of Glu³⁸⁵ in NCX1 have been reported. Given the remarkable impact of Glu⁴⁵⁵ mutations, we would predict that this Glu residue plays an important role in the Ca²⁺ regulatory mechanism of mammalian NCX1 and other exchanger proteins.

All characterized exchangers show concentration-dependent Ca²⁺ regulatory effects. For example, CALX, as seen in Fig. 5A, responds in a graded manner to the progressive administration of regulatory Ca²⁺. In contrast to NCX1-CBD2 (8, 10), CALX-CBD2 does not have Ca²⁺ binding capabilities (7). CBD1 is the only Ca²⁺ binding region within the entire Ca²⁺ regulatory domain that could be responsible for this progressive negative Ca²⁺ regulatory phenotype. Therefore, the graded Ca²⁺ regulation exhibited in Fig. 5A must represent regulatory Ca²⁺ binding at CBD1. However, with only Ca²⁺-bound structures, previously reported for CBD1 (8, 11), it is impossible to distinguish whether Ca²⁺ regulation at the level of a single exchanger protein constitutes an all-or-none versus a graded Ca²⁺ binding phenomenon.

In this study, we determined the apoform structure of CBD1 as well as that of an intermediate with two, rather than four, Ca²⁺ bound. The apoform structure clearly indicates that the primary Ca²⁺ pair is critical for the stabilization of the entire Ca²⁺ binding site of CBD1 whereas the effect of the secondary Ca²⁺ pair is quite modest. This is consistent with our functional analysis, where mutations that altered Glu⁴⁵⁵ (coordinating the primary Ca²⁺ pair) strongly disrupted Ca²⁺ regulation, whereas mutations of Glu⁵²⁰ (coordinating the secondary Ca²⁺ pair) only resulted in subtle reductions of Ca²⁺ affinity. These two Ca²⁺ pairs also interact with other carboxylate groups (Fig. 1B). Given the modest effect of the other carboxylate residues in the Ca²⁺ binding site of CBD1 suggested by a previous mutagenesis study (15), the affinity of E520A mutant (0.13 μm) or E455A (3.5 μm) should approximate the affinities for the primary Ca²⁺ pair or the secondary Ca²⁺ pair, respectively. Calcium concentrations of 0.13–3.5 μm would correspond to the reactive concentration range of regulatory Ca²⁺ for CBD1. In the dynamic Ca²⁺ environment of living cells, the primary Ca²⁺ pair with its higher affinity is expected to access CBD1 initially to establish the conformational transitions. These observations clearly elucidate that the four Ca²⁺ access the binding site of CBD1 in a sequential manner, rather than by simultaneous occupation.

The mechanism through which occupancy of the CBDs by Ca²⁺ is ultimately transduced to the transport machinery within the TM segments remains unknown. A plausible theory would be that substantial conformational changes occur upon Ca²⁺ binding, which are subsequently transmitted to the transport machinery. In fact, a fluorescent resonance energy transfer study with NCX1 indicated that Ca²⁺ binding elicits a conformational change of CBD1 (26). The apoform structure shows that CBD1 maintains the integrity of its β-sandwich conformation regardless of Ca²⁺ binding, whereas conformational changes caused by Ca²⁺ interactions are limited to the residues in the Ca²⁺ binding region as examined by CD spectroscopy and x-ray crystallography, arguing that a change of quaternary structure of CBD1 is not a requirement for the Ca²⁺ regulatory mechanism. Occupancy by the primary Ca²⁺ pair only affects the stabilization of the Ca²⁺ binding site, especially within the E–F loop, as has also been shown for NCX1-CBD1 by NMR (12). Considering the fact that the E–F loop locates to the CBD1 domain surface, we propose that the Ca²⁺ binding signal is transduced from the E–F loop by communication to adjacent domain(s).

For all NCX proteins, the C terminus of CBD1 must connect with the N terminus of CBD2 in a very compact manner, providing the strong possibility that the Ca²⁺ binding site, particularly the E–F loop of CBD1, interacts with the N-terminal region of CBD2. Our mutational analysis shows that carboxylate residues in the Ca²⁺ binding sites of CBD1 serve unequal roles in the Ca²⁺ regulatory mechanism (Fig. 5). Notably, the essential residue Glu⁴⁵⁵ locates very close to the link or hinge region, further supporting the possibility that the signal of Ca²⁺ binding is transduced through this link via domain interactions between CBD1 and CBD2. This possibility is also strongly supported by our previous mutational analysis, where a proline mutation of Gly⁵⁵⁵ completely eliminated the Ca²⁺ regulatory properties of CALX (15). This proline mutation occurs at the joint between CBD1 and CBD2 and presumably disrupts CBD domain interactions.

Overall, our results support a mechanistic hypothesis that Ca²⁺ binding/unbinding at CBD1 is transduced through the interdomain interactions between the Ca²⁺ binding sites of CBD1 and CBD2 and that the E–F loop of CBD1 acts as a hinge for this domain interaction. As a consequence, Ca²⁺ binding at CBD1 stabilizes the E–F loop and in turn changes the orientation between these two rigid domains to influence the motion, ion access, or functionality of the transmembrane segments. Given that similar effects are also reported for the analogous mutation, G503P, in NCX1 (9), this general Ca²⁺ regulatory mechanism may also be applicable to the larger Na⁺/Ca²⁺ exchanger protein family.

Based on our structural and functional analysis, we hypothesize that the Ca²⁺ regulatory mechanism occurring via CBD1 for CALX is performed in two sequential steps, as follows (Scheme 1): 1) CALX remains fully active when CBD1 exists in its apoform; 2) the primary pair of Ca²⁺ ions accesses the binding site to stabilize the E–F loop and alters the domain orientation angle between CBD1 and CBD2, generating a conformational change of the TM segments to initiate transporter inactivation; 3) as the Ca²⁺ concentration increases, the secondary Ca²⁺ pair binds to CBD1, stabilization of the E–F loop is further enhanced, and the TM transport machinery becomes more completely inactivated by stabilization of this inactive state. Notably, such notions are becoming increasingly testable based on existing structure-function studies.

SCHEME 1.

Hypothetical mechanism of Ca²⁺ regulation for the CALX Na⁺/Ca²⁺ exchanger.

The graded nature of Ca²⁺ regulatory effects are even more pronounced for NCX1 than for CALX. In NCX1, Na⁺/Ca²⁺ exchange currents are triggered by submicromolar Ca²⁺ levels and are amplified almost continuously by higher Ca²⁺ levels exceeding 10 μm, presumably in response to the much larger changes in intracellular Ca²⁺ levels that occur in cardiac myocytes. Our structures provides solid evidence that stepwise regulation of exchange is accomplished through different Ca²⁺ binding states in the CBD1 domain through its communication between the two CBDs. Very recently, a comparably functional study with NCX1 CBD1 mutants supports further application of our hypothetic model (28). Additional understanding of this interplay will need to be addressed by future crystallographic studies of the entire intracellular loop in conjunction with mutagenesis studies.