FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2028093318> ?p ?o ?g. }

• W2028093318 endingPage "21581" @default.
• W2028093318 startingPage "21577" @default.
• W2028093318 abstract "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, Asp421, and Asp500 coordinate multiple Ca2+ ions and appear to be the key residues in forming a tight binding cluster of four Ca2+ ions. Glu451 is centrally located coordinating Ca1, Ca2, and Ca3. Asp421 coordinates both Ca1 and Ca2, while Asp500 coordinates Ca3 and Ca4. These three residues appear to orient the four Ca2+ ions into a tight binding cluster. Although never previously observed, a similar arrangement of a four Ca2+ ion binding cluster has been predicted for another Ca2+ sensor domain, the C2 domains of synaptotagmin I and phospholipase C (21Ubach J. Zhang X. Shao X. Sudhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (250) Google Scholar). Two acidic segments, each characterized by three consecutive aspartic acid residues (498–500 and 446–448), were previously suggested to be Ca2+ binding regions (6Levitsky D.O. Nicoll D.A. Philipson K.D. J. Biol. Chem. 1994; 269: 22847-22852Abstract Full Text PDF PubMed Google Scholar); mutations in residues Asp447, Asp448, Asp498, and Asp500 each result in an apparent 3-fold decrease 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 mutation, E454K, showed an 8-fold decrease 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, Asp499) and three water molecules that are ligands for Ca2+, which are not part of the Ca2+ binding structure in the NMR study (9Hilge M. Aelen J. Vuister G.W. Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Conversely, 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) 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 (22Sadowski I. Stone J.C. Pawson T. Mol. Cell. Biol. 1986; 6: 4396-4408Crossref PubMed Scopus (386) Google Scholar, 23Ponting C.P. Protein Sci. 1997; 6: 464-468Crossref PubMed Scopus (193) Google Scholar). 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 (24Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. et al.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17700) Google Scholar). The binding of Ca2+ to proteins has a variety of roles. These include enhancing protein stability (25Reinhardt D.P. Ono R.N. Sakai L.Y. J. Biol. Chem. 1997; 272: 1231-1236Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 26Boggon T.J. Murray J. Chappuis-Flament S. Wong E. Gumbiner B.M. Shapiro L. Science. 2002; 296: 1308-1313Crossref PubMed Scopus (543) Google Scholar) and inducing conformational changes to facilitate secondary actions as seen with calmodulin (27Adelstein R.S. Cell. 1982; 30: 349-350Abstract Full Text PDF PubMed Google Scholar, 28Babu Y.S. Sack J.S. Greenhough T.J. Bugg C.E. Means A.R. Cook W.J. Nature. 1985; 315: 37-40Crossref PubMed Scopus (803) Google Scholar, 29Levitan I.B. Science. 2004; 304: 394-395Crossref PubMed Scopus (2) Google Scholar) and other Ca2+ sensors (30Bootman M.D. Berridge M.J. Cell. 1995; 83: 675-678Abstract Full Text PDF PubMed Scopus (393) Google Scholar). 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 (24Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. et al.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17700) Google Scholar). The majority of proteins with C2 domains are involved in signal transduction or membrane trafficking (31Rizo J. Sudhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (707) Google Scholar). The two C2 domains that are most extensively studied on a structural level are those of synaptotagmin (32Shao X. Davletov B.A. Sutton R.B. Sudhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (294) Google Scholar, 33Dai H. Shin O.H. Machius M. Tomchick D.R. Sudhof T.C. Rizo J. Nat. Struct. Mol. Biol. 2004; 11: 844-849Crossref PubMed Scopus (79) Google Scholar) and phospholipase C (34Essen L.O. Perisic O. Cheung R. Katan M. Williams R.L. Nature. 1996; 380: 595-602Crossref PubMed Scopus (516) Google Scholar), both of which form an eight-stranded β-sandwich. The β-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. The crystal structure of CBD1 reveals a new Ca2+ binding motif consisting of four Ca2+ ions arranged in a tight cluster. This coordination scheme utilizes carboxylate moieties from aspartic and glutamic acid residues to form polydendate interactions with multiple Ca2+ ions. This unique cluster facilitates the reversible binding of Ca2+ in an environment where the concentration of free Ca2+ is kept low. Further biochemical and mutational analysis based on the crystal structure and structure determinations of other components of the cytosolic loop will facilitate our understanding of the sensory mechanism of NCX. 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. Download .pdf (.04 MB) Help with pdf files" @default.
• W2028093318 created "2016-06-24" @default.
• W2028093318 creator A5007964042 @default.
• W2028093318 creator A5027136319 @default.
• W2028093318 creator A5034393915 @default.
• W2028093318 creator A5044718915 @default.
• W2028093318 creator A5053075000 @default.
• W2028093318 creator A5074384477 @default.
• W2028093318 date "2006-08-01" @default.
• W2028093318 modified "2023-10-12" @default.
• W2028093318 title "The Crystal Structure of the Primary Ca2+ Sensor of the Na+/Ca2+ Exchanger Reveals a Novel Ca2+ Binding Motif" @default.
• W2028093318 cites W1509963147 @default.
• W2028093318 cites W1539796472 @default.
• W2028093318 cites W1540755010 @default.
• W2028093318 cites W1888944045 @default.
• W2028093318 cites W1966880718 @default.
• W2028093318 cites W1969592949 @default.
• W2028093318 cites W1970692622 @default.
• W2028093318 cites W1994286350 @default.
• W2028093318 cites W1995017064 @default.
• W2028093318 cites W1997892453 @default.
• W2028093318 cites W2007745713 @default.
• W2028093318 cites W2025234475 @default.
• W2028093318 cites W2026696517 @default.
• W2028093318 cites W2030922813 @default.
• W2028093318 cites W2032781215 @default.
• W2028093318 cites W2036696283 @default.
• W2028093318 cites W2037710668 @default.
• W2028093318 cites W2038840577 @default.
• W2028093318 cites W2042055643 @default.
• W2028093318 cites W2051025707 @default.
• W2028093318 cites W2058308341 @default.
• W2028093318 cites W2061235611 @default.
• W2028093318 cites W2115291348 @default.
• W2028093318 cites W2127230663 @default.
• W2028093318 cites W2144081223 @default.
• W2028093318 cites W2145487893 @default.
• W2028093318 cites W2150487147 @default.
• W2028093318 cites W2155479906 @default.
• W2028093318 cites W2158238509 @default.
• W2028093318 cites W2168909179 @default.
• W2028093318 cites W2170680714 @default.
• W2028093318 cites W2171279562 @default.
• W2028093318 doi "https://doi.org/10.1074/jbc.c600117200" @default.
• W2028093318 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/16774926" @default.
• W2028093318 hasPublicationYear "2006" @default.
• W2028093318 type Work @default.
• W2028093318 sameAs 2028093318 @default.
• W2028093318 citedByCount "111" @default.
• W2028093318 countsByYear W20280933182012 @default.
• W2028093318 countsByYear W20280933182013 @default.
• W2028093318 countsByYear W20280933182014 @default.
• W2028093318 countsByYear W20280933182015 @default.
• W2028093318 countsByYear W20280933182016 @default.
• W2028093318 countsByYear W20280933182017 @default.
• W2028093318 countsByYear W20280933182018 @default.
• W2028093318 countsByYear W20280933182019 @default.
• W2028093318 countsByYear W20280933182020 @default.
• W2028093318 countsByYear W20280933182021 @default.
• W2028093318 countsByYear W20280933182022 @default.
• W2028093318 countsByYear W20280933182023 @default.
• W2028093318 crossrefType "journal-article" @default.
• W2028093318 hasAuthorship W2028093318A5007964042 @default.
• W2028093318 hasAuthorship W2028093318A5027136319 @default.
• W2028093318 hasAuthorship W2028093318A5034393915 @default.
• W2028093318 hasAuthorship W2028093318A5044718915 @default.
• W2028093318 hasAuthorship W2028093318A5053075000 @default.
• W2028093318 hasAuthorship W2028093318A5074384477 @default.
• W2028093318 hasBestOaLocation W20280933181 @default.
• W2028093318 hasConcept C107824862 @default.
• W2028093318 hasConcept C115624301 @default.
• W2028093318 hasConcept C145148216 @default.
• W2028093318 hasConcept C178790620 @default.
• W2028093318 hasConcept C185592680 @default.
• W2028093318 hasConcept C2777735026 @default.
• W2028093318 hasConcept C55493867 @default.
• W2028093318 hasConcept C71240020 @default.
• W2028093318 hasConcept C79879829 @default.
• W2028093318 hasConcept C8010536 @default.
• W2028093318 hasConceptScore W2028093318C107824862 @default.
• W2028093318 hasConceptScore W2028093318C115624301 @default.
• W2028093318 hasConceptScore W2028093318C145148216 @default.
• W2028093318 hasConceptScore W2028093318C178790620 @default.
• W2028093318 hasConceptScore W2028093318C185592680 @default.
• W2028093318 hasConceptScore W2028093318C2777735026 @default.
• W2028093318 hasConceptScore W2028093318C55493867 @default.
• W2028093318 hasConceptScore W2028093318C71240020 @default.
• W2028093318 hasConceptScore W2028093318C79879829 @default.
• W2028093318 hasConceptScore W2028093318C8010536 @default.
• W2028093318 hasIssue "31" @default.
• W2028093318 hasLocation W20280933181 @default.
• W2028093318 hasOpenAccess W2028093318 @default.
• W2028093318 hasPrimaryLocation W20280933181 @default.
• W2028093318 hasRelatedWork W1966292218 @default.
• W2028093318 hasRelatedWork W1966880718 @default.
• W2028093318 hasRelatedWork W1973543412 @default.
• W2028093318 hasRelatedWork W2035215415 @default.
• W2028093318 hasRelatedWork W2039482996 @default.

• next