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

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. 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. 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 (1Bers D.M. Excitation-Contraction Coupling and Cardiac Contractile Force. Kluwer, Boston2001: 133-160Google Scholar). 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 (2Ottolia M. Philipson K.D. John S. Biophys. J. 2004; 87: 899-906Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The Na+/Ca2+ exchanger protein is predicted to consist of nine transmembrane segments and a large intracellular loop (3Philipson K.D. Nicoll D.A. Ottolia M. Quednau B.D. Reuter H. John S. Qiu Z. Ann. N. Y. Acad. Sci. 2002; 976: 1-10Crossref PubMed Scopus (103) Google Scholar, 4Nicoll D.A. Ottolia M. Lu L. Lu Y. Philipson K.D. J. Biol. Chem. 1999; 274: 910-917Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). 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+ (5Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar, 6Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar). This segment comprises the first of two tandem Calx-β domains (7Schwarz E.M. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10249-10254Crossref PubMed Scopus (181) Google Scholar). Mutational analysis identified two groups of three aspartate residues within the first Calx-β domain that were associated with the binding of Ca2+ (5Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar, 6Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar). The binding of Ca2+ to the regulatory site induces substantial conformational changes that presumably mediate regulatory function (2Ottolia M. Philipson K.D. John S. Biophys. J. 2004; 87: 899-906Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 6Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar, 8Levitsky D.O. Fraysse B. Leoty C. Nicoll D.A. Philipson K.D. Mol. Cell. Biochem. 1996; 160–161: 27-32Crossref PubMed Scopus (27) Google Scholar, 9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). 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 (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Two Ca2+ binding domains (CBD1 and CBD2) were identified that correspond to Calx-β1 and -β2. 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-β 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 (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). 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. 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 β-mercaptoethanol and EDTA-free complete proteinase inhibitor (Roche Applied Science), stirred for 30 min, then sonicated. Following centrifugation at 10,000 × 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, 2The abbreviations used are: Mes, 4-morpholineethanesulfonic acid; NCX, Na+/Ca2+ exchanger; CBD, calcium binding domain. 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 (10Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38526) Google Scholar). The structure of CBD1 was phased by molecular replacement using the program PHASER (11McCoy A.J. Grosse-Kunstleve R.W. Storoni L.C. Read R.J. Acta Crystallogr Sect. D Biol. Crystallogr. 2005; 61: 458-464Crossref PubMed Scopus (1599) Google Scholar). 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 (12Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23224) Google Scholar) and refined using CNS (13Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16957) Google Scholar) and REFMAC (14Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13853) Google Scholar) with a final R and Rfree of 22.2 and 28.4%, respectively. 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 (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) 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 Cα atoms (Fig. 1). The overall positional alignment between the two structures coincides well including the notable β-bulge and cis-proline that disrupt the A and G β-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 β-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 (8Levitsky D.O. Fraysse B. Leoty C. Nicoll D.A. Philipson K.D. Mol. Cell. Biochem. 1996; 160–161: 27-32Crossref PubMed Scopus (27) Google Scholar). Hill coefficients have been variable for binding and functional effects of Ca2+. Values include 0.9 (5Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar), 1.4 (15Ottolia M. Nicoll D.A. Philipson K.D. J. Biol. Chem. 2005; 280: 1061-1069Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and 2.9 (2Ottolia M. Philipson K.D. John S. Biophys. J. 2004; 87: 899-906Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) 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 β-sandwich motif is formed by two antiparallel β-sheets consisting of strands A-B-E and strands D-C-F-G (Fig. 2a). The presence of a β-bulge in strand A disrupts the antiparallel hydrogen bonding pattern between strands A′ and B. Following the β-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 (16Halaby D.M. Poupon A. Mornon J. Protein Eng. 1999; 12: 563-571Crossref PubMed Scopus (192) Google Scholar, 17Richardson J.S. Richardson D.C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 2754-2759Crossref PubMed Scopus (654) Google Scholar) and have been suggested to be protective in preventing aggregation between multiple immunoglobulin domains by disrupting potential intermolecular hydrogen bonding surfaces (18Park S. Saven J.G. Protein Sci. 2006; 15: 200-207Crossref PubMed Scopus (16) Google Scholar). This suggestion seems particularly relevant based on the model presented by Hilge et al. (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), 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) (19Holm L. Sander C. Nucleic Acids Res. 1998; 26: 316-319Crossref PubMed Scopus (596) Google Scholar) 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 (16Halaby D.M. Poupon A. Mornon J. Protein Eng. 1999; 12: 563-571Crossref PubMed Scopus (192) Google Scholar), which is one of the most prevalent domains encoded by the human genome (20Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. et al.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10570) Google Scholar). Ca2+ Coordination—The striking difference between the crystal and NMR structures is at the Ca2+ binding region. Hilge and colleagues (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) 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 β-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. The majority of the residues involved in coordinating the Ca2+ ions are located at the C terminus (Asp498, Asp499, Asp500) and in loop E-F (Asp446, Asp447, Ile449, Glu451, Glu454). Additional interactions occur with Glu385 in the A-B loop, Asp421 in the C-D loop, and three water molecules. The overall coordination scheme for each Ca2+ site is summarized in Table 2 of the supplemental material. In short, Ca1 and Ca4 are penta-coordinated, while Ca2 and Ca3 are hexa- and hepta-coordinated, respectively. Glu451, and multiple Ca2+ ions and to be the residues in forming a tight binding cluster of four Ca2+ ions. is located coordinating and Asp421 coordinates both Ca1 and while coordinates Ca3 and These three residues to the four Ca2+ ions a tight binding cluster. Although previously a of a four Ca2+ binding cluster has been predicted for Ca2+ sensor the domains of and C J. J. J. 1998; PubMed Scopus Google Scholar). Two each by three aspartic acid residues and were previously suggested to be Ca2+ binding D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google in residues Asp447, and each in an apparent in Ca2+ affinity (2Ottolia M. Philipson K.D. John S. Biophys. J. 2004; 87: 899-906Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Additionally, a recent showed an in Ca2+ affinity (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). We directly visualize three residues (Asp446, Ile449, and three water that are for Ca2+, which are not of the Ca2+ binding structure in the NMR (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Hilge et al. (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar) as a Ca2+ but we find that residue is not directly involved in the binding of Ca2+. In the Ca2+ binding region is tightly through a coordination scheme of with Ca2+ Binding of sequence and structural data has revealed a number of protein that are and J.C. T. Mol. Cell. Biol. PubMed Scopus Google Scholar, Protein Sci. 1997; PubMed Scopus Google Scholar). These protein the regulation of that in function and impact multiple of the human genome revealed a number of Ca2+ binding L.M. B. C. J. K. K. M. W. et 2001; PubMed Scopus Google Scholar). The binding of Ca2+ to has a of These include protein J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. L. 2002; PubMed Scopus Google Scholar) and conformational changes to as with Cell. Full Text PDF PubMed Google Scholar, J.S. PubMed Scopus Google Scholar, 2004; PubMed Scopus Google Scholar) and other Ca2+ M.D. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar). CBD1 a unique binding cluster that may be by other Ca2+ sensor We sequence and structural between the CBD1 domain and the family of domains are the most Ca2+ binding present in L.M. B. C. J. K. K. M. W. et 2001; PubMed Scopus Google Scholar). The majority of with domains are involved in or membrane J. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). The two domains that are most on a structural are those of Sutton J. 1996; PubMed Scopus Google Scholar, H. M. J. Mol. Biol. 2004; PubMed Scopus Google Scholar) and C M. 1996; PubMed Scopus Google Scholar), both of which form an β-sandwich. The β-sandwich variable loops that are in the primary sequence to the binding of multiple Ca2+ ions in a cluster. to CBD1, the binding sites are primarily of aspartic acid residues forming polydentate interactions between two or three Ca2+ ions. (Fig. between CBD1 and domains a number of the first However, the of a Ca2+ as in CBD1, coordinating residues not in domain in the structure, these residues are located the C terminus in the segment but there is no structural or sequence for region in Although the CBD1 and the domains share a common Ca2+ coordination scheme that may be general for Ca2+ sensing. The crystal structure of CBD1 reveals a new Ca2+ binding motif consisting of four Ca2+ ions arranged in a tight cluster. This coordination scheme from aspartic and glutamic acid residues to form interactions with multiple Ca2+ ions. This unique cluster the binding of Ca2+ in an where the concentration of Ca2+ is Further biochemical and mutational analysis based on the crystal structure and structure of other of the loop will understanding of the sensory of We are to and of 8.2.2 of the Light Source (Berkeley, We for at the of and and for and on with