Journal of Biomolecular NMR

, Volume 54, Issue 1, pp 115–121 | Cite as

NMR structure note: solution structure of Ca2+ binding domain 2B of the third isoform of the Na+/Ca2+ exchanger

  • Vincent Breukels
  • Wouter G. Touw
  • Geerten W. Vuister
NMR structure note

Biological context

The Na+/Ca2+ exchanger (NCX) is an omnipresent plasma membrane protein that catalyzes the removal of intracellular Ca2+ for the uptake of Na+ [see for review (Lytton 2007)]. Mammals express three isoforms, denoted as NCX1, NCX2, or NCX3, of which isoforms 1 and 3 also display alternative splicing. The exchanger is activated by allosteric Ca2+ binding to Ca2+ binding domains 1 and 2 (CBD1 and CBD2) located in the large cytosolic loop between transmembrane helix 5 and 6. CBD1 binds four Ca2+ ions, irrespective of the isoform and is considered the primary Ca2+ sensor. The number of Ca2+ ions that bind to CBD2 depends on the isoform and splice variant. It is hypothesized that the number of binding sites determines the ability of the exchanger to overcome the Na+ dependent inactivation (Hilge et al. 2009) and that Mg2+ binding plays a modulating role (Boyman et al. 2009; Breukels et al. 2011).

NCX1 is thus far the best-studied isoform and solution or X-ray structures of...


Resonance Assignment Isothermal Titration Calorimetry Backbone Dynamic Alignment Medium Side Chain Chemical Shift 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Ca2+ binding domain 2


4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid


Macroscopic dissociation constant


Isothermal titration calorimetry


Na+/Ca2+ exchanger isoform 3


Nuclear Overhauser effect


Optical density at 600 nm


Residual dipolar coupling


Root mean square


Root mean square deviation


Sodium dodecyl sulfate polyacrylamide gel electrophoresis



We thank Jurgen Doreleijers for assistance with Xplor-NIH and iCing, Gjalt van Rutten for support with using the HPC Cloud infrastructure at BitBrains. This work was supported by grants from the Netherlands Organization for Scientific Research (NWO): 700.55.443 and 700.57.101.

Supplementary material

10858_2012_9654_MOESM1_ESM.pdf (3.2 mb)
Supplementary material 1 (PDF 3261 kb)


  1. Besserer GM, Ottolia M, Nicoll DA, Chaptal V, Cascio D, Philipson KD, Abramson J (2007) The second Ca2+ -binding domain of the Na+ -Ca2+ exchanger is essential for regulation: crystal structures and mutational analysis. P Natl Acad Sci USA 104:18467–18472ADSCrossRefGoogle Scholar
  2. Boyman L, Mikhasenko H, Hiller R, Khananshvili D (2009) Kinetic and equilibrium properties of regulatory calcium sensors of NCX1 protein. J Biol Chem 284(10):6185–6193CrossRefGoogle Scholar
  3. Breukels V, Vuister GW (2010) Binding of calcium is sensed structurally and dynamically throughout the second calcium-binding domain of the sodium/calcium exchanger. Proteins 78(8):1813–1824CrossRefGoogle Scholar
  4. Breukels V, Konijnenberg A, Nabuurs SM, Touw WG, Vuister GW (2011) The second Ca(2+)-binding domain of NCX1 binds Mg(2+) with high affinity. Biochemistry 50(41):8804–8812CrossRefGoogle Scholar
  5. Cornilescu G, Marquardt J, Ottiger M, Bax A (1998) Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase. J Am Chem Soc 120(27):6836–6837CrossRefGoogle Scholar
  6. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe—a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  7. Dosset P, Hus JC, Blackledge M, Marion D (2000) Efficient analysis of macromolecular rotational diffusion from heteronuclear relaxation data. J Biomol NMR 16(1):23–28CrossRefGoogle Scholar
  8. Durkin JT, Ahrens DC, Pan YC, Reeves JP (1991) Purification and amino-terminal sequence of the bovine cardiac sodium-calcium exchanger: evidence for the presence of a signal sequence. Arch Biochem Biophys 290(2):369–375CrossRefGoogle Scholar
  9. Giladi M, Sasson Y, Fang X, Hiller R, Buki T, Wang Y-X, Hirsch JA, Khananshvili D (2012) A common Ca2+-driven interdomain module governs eukaryotic NCX regulation. PLoS One 7(6):e39985Google Scholar
  10. Güntert P (2009) Automated structure determination from NMR spectra. Eur Biophys J Biophy 38(2):129–143CrossRefGoogle Scholar
  11. Hilge M, Aelen J, Vuister GW (2006) Ca2+ regulation in the Na+/Ca2+ exchanger involves two markedly different Ca2+ sensors. Mol Cell 22(1):15–25CrossRefGoogle Scholar
  12. Hilge M, Aelen J, Foarce A, Perrakis A, Vuister GW (2009) Ca2+ regulation in the Na+/Ca2+ exchanger features a dual electrostatic switch mechanism. Proc Natl Acad Sci USA 106(34):14333–14338ADSCrossRefGoogle Scholar
  13. Lytton J (2007) Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport. Biochem J 406(3):365–382CrossRefGoogle Scholar
  14. Nicoll DA, Quednau BD, Qui ZY, Xia YR, Lusis AJ, Philipson KD (1996) Cloning of a third mammalian Na+ -Ca2+ exchanger, NCX3. J Biol Chem 271(40):24914–24921CrossRefGoogle Scholar
  15. Nicoll DA, Sawaya MR, Kwon S, Cascio D, Philipson KD, Abramson J (2006) The crystal structure of the primary Ca2+ sensor of the Na+/Ca2+ exchanger reveals a novel Ca2+ binding motif. J Biol Chem 281(31):21577–21581CrossRefGoogle Scholar
  16. Salinas RK, Bruschweiler-Li L, Johnson E, Bruschweiler R (2011) Ca2+-binding alters the inter-domain flexibility between the two cytoplasmic calcium-binding domains in the Na+/Ca2+ exchanger. J Biol Chem 286(37):32123–32131Google Scholar
  17. Shen Y, Delaglio F, Cornilescu G, Bax A (2009) TALOS+ : a hybrid method for predicting protein backbone torsion angles from NMR chemical shifts. J Biomol NMR 44(4):213–223CrossRefGoogle Scholar
  18. Vranken WF, Boucher W, Stevens TJ, Fogh RH, Pajon A, Llinas P, Ulrich EL, Markley JL, Ionides J, Laue ED (2005) The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 59(4):687–696CrossRefGoogle Scholar
  19. Wu M, Wang M, Nix J, Hryshko LV, Zheng L (2009) Crystal Structure of CBD2 from the drosophila Na(+)/Ca(2+) exchanger: diversity of Ca(2+) regulation and its alternative splicing modification. J Mol Biol 387(1):104–112CrossRefGoogle Scholar
  20. Wu M, Tong S, Gonzalez J, Jayaraman V, Spudich JL, Zheng L (2011) Structural basis of the Ca(2+) inhibitory mechanism of drosophila Na(+)/Ca(2+) exchanger CALX and its modification by alternative splicing. Structure 19(10):1509–1517CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Vincent Breukels
    • 1
    • 2
  • Wouter G. Touw
    • 1
  • Geerten W. Vuister
    • 3
  1. 1.Institute for Molecules and MaterialsRadboud University NijmegenNijmegenThe Netherlands
  2. 2.Department of RadiologyRadboud University Nijmegen Medical CentreNijmegenThe Netherlands
  3. 3.Department of BiochemistryUniversity of LeicesterLeicesterUK

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