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• W1968854823 abstract "The Na+/Ca2+ exchanger CALX promotes Ca2+ efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca2+ homeostasis. CALX activity is negatively regulated by specific Ca2+ interaction within its two intracellular Ca2+ regulatory domains CBD1 and CBD2, yet how the Ca2+ binding is converted to molecular motion to operate the exchanger is unknown. Here, we report crystal structures of the entire Ca2+ regulatory domain CBD12 from two alternative splicing isoforms, CALX 1.1 and 1.2, exhibiting distinct regulatory Ca2+ dependency. The structures show an open V-shaped conformation with four Ca2+ ions bound on the CBD domain interface, confirmed by LRET analysis. The structures together with Ca2+-binding analysis support that the Ca2+ inhibition of CALX is achieved by interdomain conformational changes induced by Ca2+ binding at CBD1. The conformational difference between the two isoforms also indicates that alternative splicing adjusts the interdomain orientation angle to modify the Ca2+ regulatory property of the exchangers. The Na+/Ca2+ exchanger CALX promotes Ca2+ efflux in Drosophila sensory neuronal cells to facilitate light-mediated Ca2+ homeostasis. CALX activity is negatively regulated by specific Ca2+ interaction within its two intracellular Ca2+ regulatory domains CBD1 and CBD2, yet how the Ca2+ binding is converted to molecular motion to operate the exchanger is unknown. Here, we report crystal structures of the entire Ca2+ regulatory domain CBD12 from two alternative splicing isoforms, CALX 1.1 and 1.2, exhibiting distinct regulatory Ca2+ dependency. The structures show an open V-shaped conformation with four Ca2+ ions bound on the CBD domain interface, confirmed by LRET analysis. The structures together with Ca2+-binding analysis support that the Ca2+ inhibition of CALX is achieved by interdomain conformational changes induced by Ca2+ binding at CBD1. The conformational difference between the two isoforms also indicates that alternative splicing adjusts the interdomain orientation angle to modify the Ca2+ regulatory property of the exchangers. Crystal structures of the complete Ca2+ regulatory domain of the exchanger CALX Ca2+ binding at the domain interface between CBD1 and CBD2 Alternative splicing induces CBD domain orientation angle change Implication of the Ca2+ regulatory mechanism of the Na+/Ca2+ exchanger family Sodium-calcium exchangers (NCXs) catalyze Ca2+ flux across the plasma membrane by utilizing a counter-Na+ electrochemical gradient. NCXs play a major role in Ca2+ homeostasis in many tissues. The cardiac exchanger NCX1 facilitates cardiac contractibility on the myocytic membrane (Bers, 2001Bers D.M. Action potential & ion channels.in: Excitation-Contraction Coupling and Cardiac Contractile Force. Second Edition. Kluwer Academic Publications, Boston2001: 63-99Crossref Google Scholar); whereas NCX3 is involved in excitation-relaxation coupling in neurons (Blaustein et al., 1996Blaustein M.P. Fontana G. Rogowski R.S. The Na+-Ca2+ exchanger in rat brain synaptosomes. Kinetics and regulation.Ann. N Y Acad. Sci. 1996; 779: 300-317Crossref PubMed Scopus (35) Google Scholar). CALX, a NCX homolog found in Drosophila sensory neurons, is responsible for Ca2+ efflux after light-induced Ca2+ stimulation and the photoreceptor cell cascade (Hryshko et al., 1996Hryshko L.V. Matsuoka S. Nicoll D.A. Weiss J.N. Schwarz E.M. Benzer S. Philipson K.D. Anomalous regulation of the Drosophila Na+-Ca2+ exchanger by Ca2+.J. Gen. Physiol. 1996; 108: 67-74Crossref PubMed Scopus (69) Google Scholar, Schwarz and Benzer, 1997Schwarz E.M. Benzer S. Calx, a Na-Ca exchanger gene of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA. 1997; 94: 10249-10254Crossref PubMed Scopus (181) Google Scholar, Wang et al., 2005Wang T. Xu H. Oberwinkler J. Gu Y. Hardie R.C. Montell C. Light activation, adaptation, and cell survival functions of the Na+/Ca2+ exchanger CalX.Neuron. 2005; 45: 367-378Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). NCXs share a conserved structural model: 10 transmembrane helices (TMs) are predicted to form a Na+/Ca2+ exchange domain, along with a large intracellular regulatory region of about 500 amino acid residues between TM 5 and 6 (Figures 1A and 1B ) (Schwarz and Benzer, 1997Schwarz E.M. Benzer S. Calx, a Na-Ca exchanger gene of Drosophila melanogaster.Proc. Natl. Acad. Sci. USA. 1997; 94: 10249-10254Crossref PubMed Scopus (181) Google Scholar, Nicoll et al., 2002Nicoll D.A. Ottolia M. Philipson K.D. Toward a topological model of the NCX1 exchanger.Ann. N Y Acad. Sci. 2002; 976: 11-18Crossref PubMed Scopus (23) Google Scholar). Regulation by intracellular Ca2+ (Ca2+i) is one of the most important features of the NCX proteins (Hilgemann, 1990Hilgemann D.W. Regulation and deregulation of cardiac Na+-Ca2+ exchange in giant excised sarcolemmal membrane patches.Nature. 1990; 344: 242-245Crossref PubMed Scopus (249) Google Scholar). Two specific Ca2+-binding domains, CBD1 and CBD2, have been identified within the intracellular region (Hilge et al., 2006Hilge M. Aelen J. Vuister G.W. Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Ca2+ interaction with the two CBDs modulates Na+/Ca2+ exchange activity of NCX in response to [Ca2+i] change (Matsuoka et al., 1995Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. Regulation of the cardiac Na+-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca2+-binding domain.J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar, Besserer et al., 2007Besserer G.M. Ottolia M. Nicoll D.A. Chaptal V. Cascio D. Philipson K.D. Abramson J. The second Ca2+-binding domain of the Na+ Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.Proc. Natl. Acad. Sci. USA. 2007; 104: 18467-18472Crossref PubMed Scopus (89) Google Scholar). Despite high-sequence homology within the intracellular regions (Figure 1B), CALX exhibits a unique negative Ca2+ regulatory property in contrast to the positive effect of other characterized NCXs (Hryshko et al., 1996Hryshko L.V. Matsuoka S. Nicoll D.A. Weiss J.N. Schwarz E.M. Benzer S. Philipson K.D. Anomalous regulation of the Drosophila Na+-Ca2+ exchanger by Ca2+.J. Gen. Physiol. 1996; 108: 67-74Crossref PubMed Scopus (69) Google Scholar). Our recent X-ray crystallographic analyses of individual CBD1 and CBD2 domains from the CALX 1.1 isoform showed that only CBD1 is a functional Ca2+-binding domain (Wu et al., 2009Wu M. Wang M. Nix J. Hryshko L.V. Zheng L. Crystal structure of CBD2 from the Drosophila Na+/Ca2+ exchanger: diversity of Ca2+ regulation and its alternative splicing modification.J. Mol. Biol. 2009; 387: 104-112Crossref PubMed Scopus (40) Google Scholar, Wu et al., 2010Wu M. Le H.D. Wang M. Yurkov V. Omelchenko A. Hnatowich M. Nix J. Hryshko L.V. Zheng L. 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.J. Biol. Chem. 2010; 285: 2554-2561Crossref PubMed Scopus (32) Google Scholar). It has a nearly identical 4-Ca2+-binding conformation to that of canine NCX1 (Nicoll et al., 2006Nicoll D.A. Sawaya M.R. Kwon S. Cascio D. Philipson K.D. Abramson J. The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+ binding motif.J. Biol. Chem. 2006; 281: 21577-21581Crossref PubMed Scopus (99) Google Scholar), further supporting the importance of CBD1 in the Ca2+ regulatory mechanism of NCXs. A plausible model for the Ca2+ regulatory mechanism of CALX is that Ca2+ binding on CBD1 induces a protein conformational change in the TM domain where exchange activity resides. However, Ca2+ interaction with CBD1 from CALX or NCX1 results in a protein conformational change limited to the local Ca2+-binding site, mainly the 1E-1F loop of CBD1 (the loop between the β strands 1E and 1F), arguing that overall protein conformational change is not required for the Ca2+ regulation (Wu et al., 2010Wu M. Le H.D. Wang M. Yurkov V. Omelchenko A. Hnatowich M. Nix J. Hryshko L.V. Zheng L. 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.J. Biol. Chem. 2010; 285: 2554-2561Crossref PubMed Scopus (32) Google Scholar, Johnson et al., 2008Johnson E. Bruschweiler-Li L. Showalter S.A. Vuister G.W. Zhang F. Brüschweiler R. Structure and dynamics of Ca2+-binding domain 1 of the Na+/Ca2+ exchanger in the presence and in the absence of Ca2+.J. Mol. Biol. 2008; 377: 945-955Crossref PubMed Scopus (26) Google Scholar). NCXs have a very short linker predicted between CBD1 and CBD2 (Figure 1B). Several functional studies have suggested that CBD1-CBD2 interaction is crucial for Ca2+ regulation of the NCX proteins (Matsuoka et al., 1995Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. Regulation of the cardiac Na+-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca2+-binding domain.J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar, Dyck et al., 1998Dyck C. Maxwell K. Buchko J. Trac M. Omelchenko A. Hnatowich M. Hryshko L.V. Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites.J. Biol. Chem. 1998; 273: 12981-12987Crossref PubMed Scopus (35) Google Scholar, Giladi et al., 2010Giladi M. Boyman L. Mikhasenko H. Hiller R. Khananshvili D. Essential role of the CBD1-CBD2 linker in slow dissociation of Ca2+ from the regulatory two-domain tandem of NCX1.J. Biol. Chem. 2010; 285: 28117-28125Crossref PubMed Scopus (41) Google Scholar). Despite the fact that CBD2 of CALX is not a Ca2+-binding domain (Wu et al., 2009Wu M. Wang M. Nix J. Hryshko L.V. Zheng L. Crystal structure of CBD2 from the Drosophila Na+/Ca2+ exchanger: diversity of Ca2+ regulation and its alternative splicing modification.J. Mol. Biol. 2009; 387: 104-112Crossref PubMed Scopus (40) Google Scholar), a single mutation G555P at CBD2 near the linker region of CALX completely abolished Ca2+ regulation (Dyck et al., 1998Dyck C. Maxwell K. Buchko J. Trac M. Omelchenko A. Hnatowich M. Hryshko L.V. Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites.J. Biol. Chem. 1998; 273: 12981-12987Crossref PubMed Scopus (35) Google Scholar). The hypothesis has been raised that Ca2+ regulation of CALX is achieved through an interdomain conformational change involving both CBD1 and CBD2 (Wu et al., 2010Wu M. Le H.D. Wang M. Yurkov V. Omelchenko A. Hnatowich M. Nix J. Hryshko L.V. Zheng L. 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.J. Biol. Chem. 2010; 285: 2554-2561Crossref PubMed Scopus (32) Google Scholar). However, no structure of the complete Ca2+ regulatory CBD domain (CBD12) from any member of the exchanger family is available to date, and no structural interaction between CBDs has been deduced from individual CBD domain structures (Hilge et al., 2006Hilge M. Aelen J. Vuister G.W. Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors.Mol. Cell. 2006; 22: 15-25Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar, Besserer et al., 2007Besserer G.M. Ottolia M. Nicoll D.A. Chaptal V. Cascio D. Philipson K.D. Abramson J. The second Ca2+-binding domain of the Na+ Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis.Proc. Natl. Acad. Sci. USA. 2007; 104: 18467-18472Crossref PubMed Scopus (89) Google Scholar, Wu et al., 2009Wu M. Wang M. Nix J. Hryshko L.V. Zheng L. Crystal structure of CBD2 from the Drosophila Na+/Ca2+ exchanger: diversity of Ca2+ regulation and its alternative splicing modification.J. Mol. Biol. 2009; 387: 104-112Crossref PubMed Scopus (40) Google Scholar, Wu et al., 2010Wu M. Le H.D. Wang M. Yurkov V. Omelchenko A. Hnatowich M. Nix J. Hryshko L.V. Zheng L. 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.J. Biol. Chem. 2010; 285: 2554-2561Crossref PubMed Scopus (32) Google Scholar, Nicoll et al., 2006Nicoll D.A. Sawaya M.R. Kwon S. Cascio D. Philipson K.D. Abramson J. The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+ binding motif.J. Biol. Chem. 2006; 281: 21577-21581Crossref PubMed Scopus (99) Google Scholar). In addition our CALX CBD1 structures demonstrate three consecutive Ca2+-binding states with cumulative binding of two Ca2+ pairs (primary and secondary) in each stage (Wu et al., 2010Wu M. Le H.D. Wang M. Yurkov V. Omelchenko A. Hnatowich M. Nix J. Hryshko L.V. Zheng L. 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.J. Biol. Chem. 2010; 285: 2554-2561Crossref PubMed Scopus (32) Google Scholar). Whether successive Ca2+-binding signals are converted to responsive domain motion within the intracellular region requires the structural information of CBD12. The Ca2+ regulatory property of NCXs is also fine-tuned by alternative splicing (Dyck et al., 1999Dyck C. Omelchenko A. Elias C.L. Quednau B.D. Philipson K.D. Hnatowich M. Hryshko L.V. Ionic regulatory properties of brain and kidney splice variants of the NCX1 Na+-Ca2+ exchanger.J. Gen. Physiol. 1999; 114: 701-711Crossref PubMed Scopus (85) Google Scholar, Quednau et al., 1997Quednau B.D. Nicoll D.A. Philipson K.D. Tissue specificity and alternative splicing of the Na+/Ca2+ exchanger isoforms NCX1, NCX2, and NCX3 in rat.Am. J. Physiol. 1997; 272: C1250-C1261PubMed Google Scholar). Two splicing variants of CALX, 1.1 and 1.2, which only differ in five residues, exhibit remarkably distinct Ca2+ regulatory properties (Omelchenko et al., 1998Omelchenko A. Dyck C. Hnatowich M. Buchko J. Nicoll D.A. Philipson K.D. Hryshko L.V. Functional differences in ionic regulation between alternatively spliced isoforms of the Na+-Ca2+ exchanger from Drosophila melanogaster.J. Gen. Physiol. 1998; 111: 691-702Crossref PubMed Scopus (40) Google Scholar). CALX 1.1 shows prominently greater inhibition by regulatory Ca2+. In contrast, CALX 1.2 exhibits higher affinity for Ca2+ but much smaller inhibitory effect. The splicing region is located in a distal loop region of CBD2 (Wu et al., 2009Wu M. Wang M. Nix J. Hryshko L.V. Zheng L. Crystal structure of CBD2 from the Drosophila Na+/Ca2+ exchanger: diversity of Ca2+ regulation and its alternative splicing modification.J. Mol. Biol. 2009; 387: 104-112Crossref PubMed Scopus (40) Google Scholar). How the alternative residues on CBD2 affect the Ca2+ regulatory properties of CALX, which in fact are determined in CBD1, remains unknown. To elucidate any domain interaction between CBD1 and CBD2 and to gain structural insight into the Ca2+ regulatory mechanism of CALX and its modification by splicing, we determined the crystal structures of CBD12 containing both CBD1 and CBD2 from each of the two CALX splice variants and characterized their Ca2+-binding properties by isothermal titration calorimetry (ITC). Our data strongly suggested that the Ca2+ regulatory property of CALX and its modification by alternative splicing are mediated by interdomain conformational changes between CBD1 and CBD2. The crystal structure of the Ca2+-bound CBD12 from CALX 1.1 was first determined at 2.35 Å resolution (Table 1). In the CBD12 structure, both CBD1 and CBD2 exhibit similar immunoglobulin-like conformations with each consisting of seven β strands as seen in the individual domain structure (Figure 2A ) (Wu et al., 2009Wu M. Wang M. Nix J. Hryshko L.V. Zheng L. Crystal structure of CBD2 from the Drosophila Na+/Ca2+ exchanger: diversity of Ca2+ regulation and its alternative splicing modification.J. Mol. Biol. 2009; 387: 104-112Crossref PubMed Scopus (40) Google Scholar, Wu et al., 2010Wu M. Le H.D. Wang M. Yurkov V. Omelchenko A. Hnatowich M. Nix J. Hryshko L.V. Zheng L. 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.J. Biol. Chem. 2010; 285: 2554-2561Crossref PubMed Scopus (32) Google Scholar). Four Ca2+ ions were found in the Ca2+-binding site of CBD1 in a conformation nearly identical to that previously observed in the individual CBD1 structure (Figure 3A ) (Wu et al., 2010Wu M. Le H.D. Wang M. Yurkov V. Omelchenko A. Hnatowich M. Nix J. Hryshko L.V. Zheng L. 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.J. Biol. Chem. 2010; 285: 2554-2561Crossref PubMed Scopus (32) Google Scholar). However, CBD2 undergoes disorder at its distal region including the inactive Ca2+-binding site in the CBD12 structure, i.e., the 2E-2F loop and an additional carboxyl terminal helix CH1 are not visible in the electron density map.Table 1Data Collection and Structure Refinement StatisticsCALX1.1-CBD12CALX1.2-CBD12Data CollectionSpace groupP 43R 3Cell dimensions a, b, c (Å)62.8, 62.8, 226.3107.5, 107.5, 358.7 α, β, γ (°)90, 90, 9090, 90, 120Resolution (Å)40.0–2.35 (2.39–2.35)aOne crystal for each structure.46.2–2.9 (2.96–2.9)aOne crystal for each structure.Rsym (%)7.8 (50.2)12.3 (40.1)I /σ (I)7.7 (2.2)5.1 (1.5)Completeness (%)99.7 (99.8)91.2 (93.0)Redundancy4.6 (4.5)2.7 (2.5)RefinementResolution (Å)40.0–2.3546.2–2.9Number of unique reflections33,95434,762Rwork/Rfree (%)21.9/26.023.0/27.8Number of atoms/asu Protein3,8777,903 Ca2+816 Water4238B factors Protein51.763.3 Ca2+41.455.1 Water29.640.6Rmsd Bond lengths (Å)0.0070.008 Bond angles (°)1.1911.255Values in parentheses are for highest-resolution shell. asu, asymmetric unit.a One crystal for each structure. Open table in a new tab Figure 3Stereo Views of the Domain Interface between CBD1 and CBD2Show full caption(A) CALX 1.1.(B) CALX 1.2. CBD1, CBD2, or 2F-2G loop is depicted as a cartoon in blue, red, or green. The residues on the domain interface are shown as sticks and balls. Four Ca2+ ions (Ca1–Ca4) are drawn as green spheres. The electron density maps contoured at 1.5 σ are drawn as gray meshes.See also Figure S1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Values in parentheses are for highest-resolution shell. asu, asymmetric unit. (A) CALX 1.1. (B) CALX 1.2. CBD1, CBD2, or 2F-2G loop is depicted as a cartoon in blue, red, or green. The residues on the domain interface are shown as sticks and balls. Four Ca2+ ions (Ca1–Ca4) are drawn as green spheres. The electron density maps contoured at 1.5 σ are drawn as gray meshes. See also Figure S1. The most remarkable feature of the CBD12 structure is the domain orientation of CBD1 and CBD2. These two rigid domains are arranged in a V-shaped conformation at a 115° angle from each other around an axis at H553 (Figure 2A). The overall conformation of CBD12 is reminiscent of a “soaring eagle” with CBD1 and CBD2 forming its two open “wings.” As a result, I442 at the amino terminus of CBD1 is separated from R693 at the carboxyl terminus of CBD2 by a large distance of 70 Å. The 2F-2G loop of CBD2, where alternative splicing occurs, forms two helices (H1 and H2) nearly perpendicular to the plane of the two “wings” and lies under a hinge region as the “eagle” body (Figure 2B). One conserved short linker sequence was predicted between CBD1 and CBD2 in NCXs. In fact there is no obvious spacer between CBD1 and CBD2 in the CBD12 structure. Although D552 is involved in Ca2+ binding at CBD1, its neighboring residue H553 is considered to be part of CBD2 (Figure 3A). G555, which proline substitution abolishes Ca2+ regulation of CALX (Dyck et al., 1998Dyck C. Maxwell K. Buchko J. Trac M. Omelchenko A. Hnatowich M. Hryshko L.V. Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites.J. Biol. Chem. 1998; 273: 12981-12987Crossref PubMed Scopus (35) Google Scholar), is located on the β strand 2A of CBD2. The proximity of these residues consequently facilitates direct interaction between the two CBD domains with the Ca2+-binding site of CBD1 positioned in the central domain interface. In addition to the linker between CBD1 and CBD2, the domain interface is predominantly contributed by the Ca2+-binding site, particularly the 1E-1F loop of CBD1, which is packed in the central hinge region of CBD12 (Figure 3A). The CBD12 structure reveals the extensive interactions of this loop with other structural components. On one side four Ca2+ ions, aligned in line in the Ca2+-binding site of CBD1, stabilize the 1E-1F loop and the physical linker region with a compact Ca2+ coordination network formed by nine carboxylate residues. On another side the 1E-1F loop is structurally supported by the perpendicular H2 helix. F519 on the outer arc of the 1E-1F loop directly interacts with I674 and S678 of the H2 helix. On the top of the interface, R584 from CBD2 lays on the hinge axis to form two salt bridges with D551 and D552 and also two hydrogen bonds with D517 from the 1E-1F loop of CBD1. Four Ca2+ ions appear at different positions at the domain interface. The primary Ca2+ pair (Ca1 and Ca2) is closer to the hinge region than the secondary Ca2+ pair (Ca3 and Ca4). The former aids the assembly of the 1E-1F loop with the physical linker on the interface. To confirm that the observed open V-shaped conformation exists also in solution, we used a luminescence resonance energy transfer (LRET)-based assay to investigate the protein conformation without crystallization constraints. The N-terminal His tag-associated Cy3 derivative of nitrilotriacetic acid chelate of nickel ((Ni-NTA)2Cy3) of the protein served as the acceptor, and three individual cysteine mutations representing positions S581, T560, and T567 (Figure 2A) were made in CBD2 to allow the introduction of a terbium chelate donor fluorophore to probe relative distances using lifetime LRET measurements. There was no difference in fluorescence signal lifetime among the three cysteine mutants without the acceptor probe (Figure 4A ), indicating no significant perturbation of the structure from the cysteine mutations. Therefore, lifetimes indicating the extent of energy transfer at different donor positions in the donor-acceptor labeled proteins can be used to estimate relative donor distances from the acceptor (Figure 4B and Table 2). These measurements confirmed that the distance from the approximate N-terminal His tag to the C terminus (T567) is significantly longer than to the hinge region (S581) and to the middle point (T560) of CBD2, as predicted from the CBD12 structure. Notably, the distance differences between LRET measurements and structural observations can be attributed to the extra N-terminal spacer sequence before the acceptor. We conclude that the open V-shaped conformation observed in our crystal structures exists also in solution and, therefore, probably also represents the physiological condition of CALX on the cell membrane.Table 2LRET Lifetimes and Calculated DistancesDonor Position581560567Donor lifetime (μs)1607 ± 551607 ± 691612 ± 38Sensitized emission lifetime (μs)81 ± 277 ± 4380 ± 32Distance (LRET) (Å)42 ± 241 ± 256 ± 5Distance in crystal structure (Å)aDistances are measured between the Cα atoms between the N-terminal I422 and respective donor residues.445370a Distances are measured between the Cα atoms between the N-terminal I422 and respective donor residues. Open table in a new tab The two CALX-splicing variants 1.1 and 1.2 have five residue differences within the region (651–655) (Figure 5A ). These five residues are located within a loop between the H1 helix and the β strand 2F of CBD2 (Figure 1B). Despite the clearly resolved conformation of this region, this splicing region has no direct contact with the Ca2+-binding site of CBD1 (Figure 5B). The molecular mechanism for alternative splicing modification of CALX could not be deduced from the CALX 1.1 structure. In addition both CBD12 proteins from the two isoforms exhibit similar monophasic Ca2+-binding profiles with similar binding affinities (160 ± 35 nM for CALX 1.1, 210 ± 38 nM for CALX 1.2), determined by ITC measurements (Figures 6A and 6B ), indicating no direct impact of splicing alternation on the Ca2+-binding property of CBD12.Figure 6Ca2+-Binding Assays of CBD12 and Mutants from CALX by ITCShow full caption(A) CALX 1.1 CBD12 wild-type.(B) CALX 1.2 CBD12 wild-type.(C) F519A.(D) I674Y.(E) S678Y.(F) H553P.(G) G555P.(H) R584A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) CALX 1.1 CBD12 wild-type. (B) CALX 1.2 CBD12 wild-type. (C) F519A. (D) I674Y. (E) S678Y. (F) H553P. (G) G555P. (H) R584A. To demonstrate the structural modification of the CBD12 conformation by alternative splicing, we determined the crystal structure of CBD12 from the splicing variant CALX 1.2 at 2.9 Å resolution (Table 1). The CBD12 structure of CALX 1.2 shows a similar V-shaped conformation as seen in the CALX 1.1 structure. CBD1 and CBD2 are also identical to those in CALX 1.1, and four Ca2+ ions were bound at the same Ca2+-binding site as seen in CALX 1.1 (Figure 3B). However, the angle between the CBD1 and CBD2 “wings” undergoes a considerable change. By superimposing the invariant CBD1 of the two isoform structures, the CBD2 of CALX 1.2 rotates along the hinge axis of H553 upward by 9° and results in a movement of the C-terminal R693 by 6.6 Å (Figure 5A). This movement is clearly attributable to the alternative splicing region. In CALX 1.1 the five alternative residues constitute a 310 end of H1 (Figure 5B). The carbonyl group of D651 forms a hydrogen bond with the amide group of A654 to terminate the H1 helix. Meanwhile, its carboxyl group interacts with D649 to stabilize the P648-induced turn conformation. In sharp contrast the alternative residues extend the H1 helix by one additional turn toward the 2B-2C loop of CBD2 in CALX 1.2 (Figure 5C). The interaction is reinforced by two hydrogen bond formations of A648 and S651 with T586 on the 2B-2C loop of CBD2. The interface between H2 and the 1E-1F loop of CBD1 remains unaffected. Thus, the conformational changes occurring on the loop between the H1 helix and the β strand body of CBD2 eventually lead the rigid CBD2 domain to undergo a closure rotation around the hinge. The splicing region in CALX 1.2 is closer to the Ca2+-binding site; one of the alternative residues, H653, interacts with the main chain of D517 on the 1E-1F loop of CBD1. Notably, none of the splicing region residues is involved in crystal contacts (see Figure S1 available online). The carboxylate residues in the Ca2+-binding site of CBD1 have been extensively studied (Matsuoka et al., 1995Matsuoka S. Nicoll D.A. Hryshko L.V. Levitsky D.O. Weiss J.N. Philipson K.D. Regulation of the cardiac Na+-Ca2+ exchanger by Ca2+. Mutational analysis of the Ca2+-binding domain.J. Gen. Physiol. 1995; 105: 403-420Crossref PubMed Scopus (204) Google Scholar, Wu et al., 2010Wu M. Le H.D. Wang M. Yurkov V. Omelchenko A. Hnatowich M. Nix J. Hryshko L.V. Zheng L. 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.J. Biol. Chem. 2010; 285: 2554-2561Crossref PubMed Scopus (32) Google Scholar, Dyck et al., 1998Dyck C. Maxwell K. Buchko J. Trac M. Omelchenko A. Hnatowich M. Hryshko L.V. Structure-function analysis of CALX1.1, a Na+-Ca2+ exchanger from Drosophila. Mutagenesis of ionic regulatory sites.J. Biol. Chem. 1998; 273: 12981-12987Crossref PubMed Scopus (35) Google Scholar). In the CBD12 structures the Ca2+-bound 1E-1F loop is a key component of the domain interface conformation (Figure 3). To investigate crosstalk between Ca2+ binding and the interface, we mutated these residues on the domain interface and evaluated the Ca2+-binding properties of the mutant proteins by ITC. Despite the fact that these residues are not directly involved in Ca2+ binding, perturbations of the 1E-1F loop interface remarkably alter the Ca2+-binding profile. F519A exhibits a biphasic Ca2+-binding curve instead of the monophasic one characteristic of wild-type. In addition, saturation of Ca2+ binding was shifted to a higher [Ca2+] (Figure 6C). We also mutated I674 or S678, the interface partners of F519 on the H2 helix of CBD2, to bulky tyrosine residues. Both mutants exhibited a similar biphasic curve as that of F519A (Figures 6D and 6E), suggesting that the interface is important for the conformation of the Ca2+-binding site. To explore the physical linker region between CBD1 and CBD2, we mutated the hinge residue H553 to proline. Although no notable difference from the wild-type was observed (Figure 6F), the trans-configuration of proline at the hinge region may favor the V-shaped conformation. Interestingly, the G555P mutant showed a near-biphasic titration curve with 10-fo" @default.
• W1968854823 created "2016-06-24" @default.
• W1968854823 creator A5023492554 @default.
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• W1968854823 date "2011-10-01" @default.
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• W1968854823 title "Structural Basis of the Ca2+ Inhibitory Mechanism of Drosophila Na+/Ca2+ Exchanger CALX and Its Modification by Alternative Splicing" @default.
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• W1968854823 doi "https://doi.org/10.1016/j.str.2011.07.008" @default.
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