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J. Biol. Chem., Vol. 281, Issue 31, 21577-21581, August 4, 2006

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The Crystal Structure of the Primary Ca²⁺ Sensor of the Na⁺/Ca²⁺ Exchanger Reveals a Novel Ca²⁺ Binding Motif^*

Debora A. Nicoll, Michael R. Sawaya, Seunghyug Kwon, Duilio Cascio, Kenneth D. Philipson, and Jeff Abramson¹

From the Department of Physiology and the Cardiovascular Research Laboratories, David Geffen School of Medicine, University of California, Los Angeles, California 90095 and the Department of Chemistry and Biochemistry, UCLA-Department of Energy Center for Genomics and Proteomics, Molecular Biology Institute, University of California, Los Angeles, California 90095

Received for publication, May 15, 2006

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
The Na⁺/Ca²⁺ exchanger is a plasma membrane protein that regulates intracellular Ca²⁺ levels in cardiac myocytes. Transport activity is governed by Ca²⁺, and the primary Ca²⁺ sensor (CBD1) is located in a large cytoplasmic loop connecting two transmembrane helices. The binding of Ca²⁺ to the CBD1 sensory domain results in conformational changes that stimulate the exchanger to extrude Ca²⁺. Here, we present a crystal structure of CBD1 at 2.5Å resolution, which reveals a novel Ca²⁺ binding site consisting of four Ca²⁺ ions arranged in a tight planar cluster. This intricate coordination pattern for a Ca²⁺ binding cluster is indicative of a highly sensitive Ca²⁺ sensor and may represent a general platform for Ca²⁺ sensing.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Rapid fluxes of Ca²⁺ across the sarcolemmal membrane are an important component of cardiac excitation-contraction coupling. Ca²⁺ influx mediated by voltage-dependent Ca²⁺ channels initiates contractions, while Ca²⁺ efflux is dominated by the Na⁺/Ca²⁺ exchanger (1). Thus, the Na⁺/Ca²⁺ exchanger is an important component of regulation of cardiac contractility. Under most physiological conditions, the exchanger uses the energy stored in the inwardly directed Na⁺ gradient to catalyze the extrusion of Ca²⁺ from the cell with a stoichiometry of 3 Na⁺ for 1 Ca²⁺.

Activity of the Na⁺/Ca²⁺ exchanger is modulated by the binding of Ca²⁺ to a high affinity regulatory site on an intracellular portion of the protein. Regulatory Ca²⁺ is not transported but potently activates exchange activity. Recent evidence suggests that Ca²⁺ may bind to and dissociate from its regulatory site during the rapid Ca²⁺ fluctuations that occur during a cardiac contraction cycle (2). The Na⁺/Ca²⁺ exchanger protein is predicted to consist of nine transmembrane segments and a large intracellular loop (3, 4). The transmembrane segments translocate ions across the membrane, and the intracellular loop is largely responsible for regulation of activity. We have previously identified a region of the intracellular loop of the exchanger (amino acids 371–508) that binds Ca²⁺ with high affinity and mediates activation of exchange activity by Ca²⁺ (5, 6). This segment comprises the first of two tandem Calx- domains (7). Mutational analysis identified two groups of three aspartate residues within the first Calx- domain that were associated with the binding of Ca²⁺ (5, 6). The binding of Ca²⁺ to the regulatory site induces substantial conformational changes that presumably mediate regulatory function (2, 6, 8, 9).

A recent major development in the understanding of Ca²⁺ regulation has been the determination of the structure of the Ca²⁺ binding region of the large intracellular loop using NMR techniques (9). Two Ca²⁺ binding domains (CBD1 and CBD2) were identified that correspond to Calx-1 and -2. CBD1 encompasses the same region that we had identified as being responsible for Ca²⁺ regulation. Binding of Ca²⁺ to CBD1 induces a substantial conformational change consistent with earlier studies. In the presence of Ca²⁺, both CBD1 and CBD2 have an immunoglobulin fold. CBD2, in the adjoining Calx- repeat region, binds Ca²⁺ with substantially lower affinity and its functional role is unclear. Unlike CBD1, the removal of Ca²⁺ from CBD2 does not induce protein unfolding.

The NMR structure of CBD1 shows a classical immunoglobulin fold with two Ca²⁺ ions bound in the distal loops (9). However, the heteronuclear single quantum correlation spectra employed by Hilge and colleagues does not directly visualize the presence of Ca²⁺ but rather infers positions from Yb³⁺-induced shifts. Here, we describe the crystal structure of CBD1 using x-ray techniques. Like the NMR structure, we find an immunoglobulin fold, and the two structures superimpose well. Strikingly, the x-ray structure reveals the presence of four Ca²⁺ ions bound in a unique cluster with important physiological consequences.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
Expression and Purification of CBD1—A fusion protein encoding residues 370–509 of Na⁺/Ca²⁺ exchanger (NCX) with an N-terminal extension of MRGSHHHHHHGI was expressed using the pQE32 vector (Qiagen) and M15pRep4 Escherichia coli cells (Qiagen). Induced cell pellets were dissolved in buffer B (8 M urea, 20 mM Tris-Cl, pH 8, 0.1 mM CaCl₂, 300 mM NaCl) supplemented with 5 mM -mercaptoethanol and EDTA-free complete proteinase inhibitor (Roche Applied Science), stirred for 30 min, then sonicated. Following centrifugation at 10,000 x g for 45 min the supernatant was filtered, Triton X-100 (1%), imidazole (10 mM), and nickel-nitrilotriacetic acid (Qiagen) were added and swirled for 30 min before loading in to a column. The column was washed with buffer B followed by washes with 75% buffer B/25% wash buffer (250 mM Mes,² pH 6.3, 0.3 M NaCl, 10% glycerol, 0.1 mM CaCl₂), 50% buffer B/50% wash buffer followed by 25% buffer B/75% wash buffer and finally with 100% wash buffer. Fusion protein was eluted from the column in wash buffer + 250 mM imidazole, pH 7.4. Fractions containing CBD1 were pooled and concentrated with Centriprep 30 (Amicon) filtered and applied to a HiPrep 16/60 Sephacryl S-100 column (Amersham Biosciences) preequilibrated with wash buffer 2 (20 mM Tris-Cl, pH 8, 300 mM NaCl, 0.1 mM CaCl₂). Peak fractions were pooled and dialyzed against five changes of 10 mM Tris-Cl, pH 7.4, 0.2 mM EGTA. Dialyzed protein was concentrated with Centricon 30 (Amicon).

Crystal Growth and Structure Determination—Purified CBD1 protein was maintained in a solution of 10 mM Tris-HCl, pH = 7.4, + 0.2 mM EGTA at a concentration of 25 mg/ml. This solution was screened against 480 commercially available crystallization conditions with the mosquito crystallization robot (TTP Labtech) using the hanging drop vapor diffusion technique. Crystals were obtained at 20 °C in condition number 35 of Hampton Research's Crystal Screen 2 (100 mM HEPES, pH = 7.5, + 70% 2-methylpentane-2,3-diol). These crystals were then optimized by addition of 100 mM guanidine discovered through additive screening using Hampton Research's Additive Screen in conjunction with the Mosquito robot. The resulting crystals diffracted to 2.5 Å resolution (see Table 1 of supplemental material).

Data were collected from a cryo-cooled crystal at beamline 8.2.2 of the Advance Light Source (Berkeley, CA). The crystal belongs to the space group P2₁2₁2 with cell dimensions of a = 59.6 Å, b = 45.5 Å, and c = 57.3 Å. Image data were processed using the programs DENZO and SCALEPACK (10). The structure of CBD1 was phased by molecular replacement using the program PHASER (11). The coordinates of the recent NMR structure of CBD1 (PDB accession code 2FWS) were used for the search model. The structure was built using the program COOT (12) and refined using CNS (13) and REFMAC (14) with a final R and R_free of 22.2 and 28.4%, respectively.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 
We sought to uncover the principles underlying Ca²⁺ regulation of the NCX by resolving the crystal structures of the primary Ca²⁺ binding domain (CBD1) in the Ca²⁺-bound and Ca²⁺-free conformations. Initial crystallization trials in the presence of 2 mM CaCl₂ (Ca²⁺-bound) and 2 mM EGTA (Ca²⁺-free) failed. To minimize the impact of these reagents on crystallization, we reduced their concentrations to 0.2 mM. An EGTA-containing sample yielded crystals diffracting to 2.5 Å. The crystal structure had a strong resemblance to the NMR structure (9) maintaining the overall immunoglobulin fold. In addition, the positions of four tightly clustered Ca²⁺ ions were revealed. Further analysis confirmed a contamination of 0.12 mM Ca²⁺ in condition number 35 of Hampton Research's Crystal Screen 2, which inadvertently led to the Ca²⁺-bound structure.

Structure Overview—The NMR and crystal structures were superimposed with a root mean square difference of 1.8 for 128 C atoms (Fig. 1). The overall positional alignment between the two structures coincides well including the notable -bulge and cis-proline that disrupt the A and G -strands, respectively. The striking new feature of the crystal structure is the presence of a novel Ca²⁺ binding site situated in the distal loops of the -sandwich containing four Ca²⁺ ions coordinated by an extensive network of amino acids residues. The previously reported NMR structure showed two Ca²⁺ ions, which approximately represent a positional average of those observed in the crystal structure (Fig. 1). This newly observed Ca²⁺ binding motif was only revealed by x-ray crystallography and will provide a framework for further biochemical and mutational analysis.

There had not previously been any indication that four Ca²⁺ ions were present in the Ca²⁺ regulatory domain. Ca²⁺ binding data had suggested the binding of two Ca²⁺ ions per regulatory domain (8). Hill coefficients have been variable for binding and functional effects of Ca²⁺. Values include 0.9 (5), 1.4 (15), and 2.9 (2) consistent with the involvement of multiple Ca²⁺ ions, although the source of the variability is unclear.

CBD1 is arranged in a classical immunoglobulin fold, where the -sandwich motif is formed by two antiparallel -sheets consisting of strands A-B-E and strands D-C-F-G (Fig. 2a). The presence of a -bulge in strand A disrupts the antiparallel hydrogen bonding pattern between strands A' and B. Following the -bulge, strand A' associates with strand G' from the opposing sheet, rather than resuming its interactions with strand B (Fig. 2c). Additionally, there is a cis-proline residue that induces an abrupt loop in the middle of strand G, but unlike strand A, strand G resumes a normal hydrogen bonding pattern with strand F. These geometrical distortions are often observed in external strands A and G of immunoglobulin folds (16, 17) and have been suggested to be protective in preventing aggregation between multiple immunoglobulin domains by disrupting potential intermolecular hydrogen bonding surfaces (18). This suggestion seems particularly relevant based on the model presented by Hilge et al. (9), predicting that the high affinity Ca²⁺ sensor (CBD1) and the low affinity Ca²⁺ sensor (CBD2) form a heterodimer stacked along the A-G interface.

The coordinates for CBD1 were compared against other three-dimensional structures using the distance matrix alignment server (Dali) (19) revealing a number of structural homologues including fibronectins, cadherins, and integrins. Although there appears to be no apparent sequence identity or functional similarities, members of the immunoglobulin fold family share a common core structure (16), which is one of the most prevalent domains encoded by the human genome (20).

Ca²⁺ Coordination—The striking difference between the crystal and NMR structures is at the Ca²⁺ binding region. Hilge and colleagues (9) were able to assign the positions for two Ca²⁺ ions by using a three prong approach, which included the recording of pseudo-contact shift data, obtaining spectra from the sample in the presence of Yb³⁺ ions and utilizing biochemical and mutagenesis data for distance constraints. However, the crystal structure revealed an extensive coordination scheme connecting four Ca²⁺ ions clustered in the distal loops of the -sandwich. It appears that the two Ca²⁺ sites predicted in the NMR structure represent a positional average of those observed in the crystal structure (Fig. 1). The four binding sites are arranged in a parallelogram-like configuration, where the distances between Ca²⁺ sites 1 and 2, 2 and 3, and 3 and 4 are 4.27, 4.30, and 3.93 Å, respectively (Fig. 2, a and b). These binding sites are primarily coordinated by aspartic and glutamic acid residues forming polydentate interactions, often between two or three Ca²⁺ ions.

View larger version (32K): [in this window] [in a new window]   FIGURE 1. Structural alignment of the crystal and NMR structures of CBD1. The NMR structure (yellow) was superimposed onto the crystal structure (blue) with a root mean square deviation of 1.8 Å for 128 C atoms. The Ca2+ ions are represented as spheres maintaining the same color code. The two Ca2+ ions from the NMR structure (yellow) seem to represent a positional average of the four Ca2+ ions seen in the crystal structure (blue).

Two acidic segments, each characterized by three consecutive aspartic acid residues (498–500 and 446–448), were previously suggested to be Ca²⁺ binding regions (6); mutations in residues Asp⁴⁴⁷, Asp⁴⁴⁸, Asp⁴⁹⁸, and Asp⁵⁰⁰ each result in an apparent 3-fold decrease in Ca²⁺ affinity (2). Additionally, a recent mutation, E454K, showed an 8-fold decrease in Ca²⁺ affinity (9). We directly visualize three residues (Asp⁴⁴⁶, Ile⁴⁴⁹, Asp⁴⁹⁹) and three water molecules that are ligands for Ca²⁺, which are not part of the Ca²⁺ binding structure in the NMR study (9). Conversely, Hilge et al. (9) place Asp⁴⁴⁸ as a Ca²⁺ ligand, but we find that this residue is not directly involved in the binding of Ca²⁺. In total, the Ca²⁺ binding region is tightly regulated through a complex coordination scheme composed mostly of carboxylate moieties.

Comparison with Other Ca²⁺ Binding Proteins—Analysis of sequence and structural data has revealed a number of protein modules that are widespread and repeated throughout nature (22, 23). These protein modules facilitate the regulation of numerous proteins that vary dramatically in function and impact multiple cellular processes. Analysis of the human genome revealed a number of Ca²⁺ binding modules (24). The binding of Ca²⁺ to proteins has a variety of roles. These include enhancing protein stability (25, 26) and inducing conformational changes to facilitate secondary actions as seen with calmodulin (27–29) and other Ca²⁺ sensors (30). CBD1 forms a unique binding cluster that may be utilized by other Ca²⁺ sensor proteins.

We note sequence and structural similarities between the CBD1 domain and the larger family of C₂ domains. C₂ domains are the second most abundant Ca²⁺ binding module present in nature (24). The majority of proteins with C₂ domains are involved in signal transduction or membrane trafficking (31). The two C₂ domains that are most extensively studied on a structural level are those of synaptotagmin (32, 33) and phospholipase C (34), both of which form an eight-stranded -sandwich. The -sandwich scaffold permits variable loops that are widely separated in the primary sequence to facilitate the binding of multiple Ca²⁺ ions in a cluster. Similar to CBD1, the binding sites are comprised primarily of aspartic acid residues forming polydentate interactions between two or three Ca²⁺ ions. Sequence alignments (Fig. 3) between CBD1 and C₂ domains show a number of similarities around the first acidic segment. However, the existence of a fourth Ca²⁺ site, as found in CBD1, would require additional acidic coordinating residues not seen in C₂ domain structures. As seen in the current structure, these additional residues are located toward the C terminus in the second acidic segment but there is no structural or sequence similarity for this region in C₂ domains. Although functionally diverse, the CBD1 and the C₂ domains share a common Ca²⁺ coordination scheme that may be general for Ca²⁺ sensing.

View larger version (43K): [in this window] [in a new window]   FIGURE 2. Structure of CBD1. a, ribbon representation of CBD1. The seven -strands are colored from the N terminus (N) in blue to the C terminus (C) in red in the same orientation as in Fig. 1. The four Ca2+ ions are depicted as green spheres. b, stereo view of the Ca2+ binding sites. The main chain is represented in blue. The four Ca2+ ions and three water molecules are colored as green and red spheres, respectively. The side chain carbons and oxygens are yellow and red, respectively. Coordination to the Ca2+ ions is represented by black dashed lines. c, secondary structure schematic of CBD1. The -strands are depicted as blue arrows labeled from A to G, and the residues involved in Ca2+ binding are shown in red circles. The A and G strands are disrupted by a -bulge and a cis-proline, respectively, resulting in strands A' and G'. The red box around A' indicates a break in hydrogen bonding arrangement, which results in a parallel alignment with strand G'.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Sequence alignment between CBD1 and a representative set of C2 domains positioned around the two acidic segments. The two acidic segments located in CBD1 are underlined in green, and the residues coordinating Ca2+ are shown in yellow background. Residues whose backbone carbonyl groups coordinate Ca2+ ions are shown on a blue background. The blue arrows represent -strands E-F-G (CBD1), 6-7-8 (SYN1) and 5-6-7 (PLC1). CBD1 is the sequence of the primary Ca2+ binding sensor of NCX (canine); SYNI is the sequence of the C2A domain of synaptotagmin I (rat); PLCI is the C2 domain of phospholipase C (rat).

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant HL-49101 (to K. D. P.) and American Heart Association Grant 0630258N (to J. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables 1 and 2. The atomic coordinates and structure factors (code 2DPK) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

¹ To whom correspondence should be addressed: Dept. of Physiology, David Geffen School of Medicine, University of California, Los Angeles, CA 90095. Tel.: 310-825-3913; Fax: 310-206-5661; E-mail: jabramson{at}mednet.ucla.edu.

² The abbreviations used are: Mes, 4-morpholineethanesulfonic acid; NCX, Na⁺/Ca²⁺ exchanger; CBD, calcium binding domain.

	ACKNOWLEDGMENTS

 
We are grateful to Corie Ralston and all personnel of beam line 8.2.2 of the Advanced Light Source (Berkeley, California). We thank Elon Hartman for contributions at the early stage of this work and Rachna Ujwal and Gabriel Mercado for helpful discussion and continual support on this project.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

 

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