Structure, binding interface and hydrophobic transitions of Ca2+-loaded calbindin-D28K

Nature Structural & Molecular Biology volume 13, pages641–647(2006)Cite this article

Abstract

Calbindin-D28K is a Ca2+-binding protein, performing roles as both a calcium buffer and calcium sensor. The NMR solution structure of Ca2+-loaded calbindin-D28K reveals a single, globular fold consisting of six distinct EF-hand subdomains, which coordinate Ca2+ in loops on EF1, EF3, EF4 and EF5. Target peptides from Ran-binding protein M and myo-inositol monophosphatase, along with a new target from procaspase-3, are shown to interact with the protein on a surface comprised of α5 (EF3), α8 (EF4) and the EF2-EF3 and EF4-EF5 loops. Fluorescence experiments reveal that calbindin-D28K adopts discrete hydrophobic states as it binds Ca2+. The structure, binding interface and hydrophobic characteristics of Ca2+-loaded calbindin-D28K provide the first detailed insights into how this essential protein may function. This structure is one of the largest high-resolution NMR structures and the largest monomeric EF-hand protein to be solved to date.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

Rent or Buy

All prices are NET prices.

Figure 1: NMR structure ensemble.
Figure 2: Long-distance EF-hand pair-pair interactions.
Figure 3: Peptide titrations of Ca2+-loaded calbindin-D28K and binding interface.

Accession codes

Primary accessions

Protein Data Bank

References

  1. 1

    Gross, M. & Kumar, R. Physiology and biochemistry of vitamin D-dependent calcium binding proteins. Am. J. Physiol. 259, F195–F209 (1990).

    CAS  PubMed  Google Scholar 

  2. 2

    Oberholtzer, J.C., Buettger, C., Summers, M.C. & Matschinsky, F.M. The 28-kDa calbindin-D is a major calcium-binding protein in the basilar papilla of the chick. Proc. Natl. Acad. Sci. USA 85, 3387–3390 (1988).

    CAS  Article  Google Scholar 

  3. 3

    Lutz, W. et al. Calbindin D28K interacts with Ran-binding protein M: identification of interacting domains by NMR spectroscopy. Biochem. Biophys. Res. Commun. 303, 1186–1192 (2003).

    CAS  Article  Google Scholar 

  4. 4

    Berggard, T., Szczepankiewicz, O., Thulin, E. & Linse, S. Myo-inositol monophosphatase is an activated target of calbindin D28K . J. Biol. Chem. 277, 41954–41959 (2002).

    CAS  Article  Google Scholar 

  5. 5

    Christakos, S. & Liu, Y. Biological actions and mechanism of action of calbindin in the process of apoptosis. J. Steroid Biochem. Mol. Biol. 89–90, 401–404 (2004).

    Article  Google Scholar 

  6. 6

    Berggard, T., Thulin, E., Akerfeldt, K.S. & Linse, S. Fragment complementation of calbindin D28K . Protein Sci. 9, 2094–2108 (2000).

    CAS  Article  Google Scholar 

  7. 7

    Linse, S. et al. Domain organization of calbindin D28K as determined from the association of six synthetic EF-hand fragments. Protein Sci. 6, 2385–2396 (1997).

    CAS  Article  Google Scholar 

  8. 8

    Bellido, T., Huening, M., Raval-Pandya, M., Manolagas, S.C. & Christakos, S. Calbindin D28K is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity. J. Biol. Chem. 275, 26328–26332 (2000).

    CAS  Article  Google Scholar 

  9. 9

    Rabinovitch, A., Suarez-Pinzon, W.L., Sooy, K., Strynadka, K. & Christakos, S. Expression of calbindin- D28K in a pancreatic islet beta-cell line protects against cytokine-induced apoptosis and necrosis. Endocrinology 142, 3649–3655 (2001).

    CAS  Article  Google Scholar 

  10. 10

    Liu, Y. et al. Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin- D28K . J. Bone Miner. Res. 19, 479–490 (2004).

    CAS  Article  Google Scholar 

  11. 11

    Christakos, S. et al. Vitamin D target proteins: function and regulation. J. Cell. Biochem. 88, 238–244 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Shamir, A., Elhadad, N., Belmaker, R.H. & Agam, G. Interaction of calbindin D28K and inositol monophosphatase in human postmortem cortex: possible implications for bipolar disorder. Bipolar Disord. 7, 42–48 (2005).

    CAS  Article  Google Scholar 

  13. 13

    Schmidt, H., Schwaller, B. & Eilers, J. Calbindin D28K targets myo-inositol monophosphatase in spines and dendrites of cerebellar Purkinje neurons. Proc. Natl. Acad. Sci. USA 102, 5850–5855 (2005).

    CAS  Article  Google Scholar 

  14. 14

    Venters, R.A. et al. The effects of Ca2+ binding on the conformation of calbindin D28K: a nuclear magnetic resonance and microelectrospray mass spectrometry study. Anal. Biochem. 317, 59–66 (2003).

    CAS  Article  Google Scholar 

  15. 15

    Morgan, D.W., Welton, A.F., Heick, A.E. & Christakos, S. Specific in vitro activation of Ca,Mg-ATPase by vitamin D-dependent rat renal calcium binding protein (calbindin D28K). Biochem. Biophys. Res. Commun. 138, 547–553 (1986).

    CAS  Article  Google Scholar 

  16. 16

    Reisner, P.D., Christakos, S. & Vanaman, T.C. In vitro enzyme activation with calbindin- D28K, the vitamin D-dependent 28 kDa calcium binding protein. FEBS Lett. 297, 127–131 (1992).

    CAS  Article  Google Scholar 

  17. 17

    Ikura, M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem. Sci. 21, 14–17 (1996).

    CAS  Article  Google Scholar 

  18. 18

    Nakamura, M. et al. When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin. J. Cell Biol. 143, 1041–1052 (1998).

    CAS  Article  Google Scholar 

  19. 19

    Nishimoto, T. A new role of ran GTPase. Biochem. Biophys. Res. Commun. 262, 571–574 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Nishitani, H. et al. Full-sized RanBPM cDNA encodes a protein possessing a long stretch of proline and glutamine within the N-terminal region, comprising a large protein complex. Gene 272, 25–33 (2001).

    CAS  Article  Google Scholar 

  21. 21

    Rao, M.A. et al. RanBPM, a nuclear protein that interacts with and regulates transcriptional activity of androgen receptor and glucocorticoid receptor. J. Biol. Chem. 277, 48020–48027 (2002).

    CAS  Article  Google Scholar 

  22. 22

    Seki, T., Hayashi, N. & Nishimoto, T. RCC1 in the Ran pathway. J. Biochem. 120, 207–214 (1996).

    CAS  Article  Google Scholar 

  23. 23

    Moore, M.S. Generation of GTP-Ran for nuclear protein import. Science 272, 47 (1996).

    CAS  Article  Google Scholar 

  24. 24

    Cotman, C.W., Poon, W.W., Rissman, R.A. & Blurton-Jones, M. The role of caspase cleavage of tau in Alzheimer disease neuropathology. J. Neuropathol. Exp. Neurol. 64, 104–112 (2005).

    CAS  Article  Google Scholar 

  25. 25

    Hodges, A. et al. Regional and cellular gene expression changes in human Huntington's disease brain. Hum. Mol. Genet. 15, 965–977 (2006).

    CAS  Article  Google Scholar 

  26. 26

    Wellington, C.L. et al. Inhibiting caspase cleavage of huntingtin reduces toxicity and aggregate formation in neuronal and nonneuronal cells. J. Biol. Chem. 275, 19831–19838 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Yuan, J. & Yankner, B.A. Apoptosis in the nervous system. Nature 407, 802–809 (2000).

    CAS  Article  Google Scholar 

  28. 28

    Lu, T. et al. Gene regulation and DNA damage in the ageing human brain. Nature 429, 883–891 (2004).

    CAS  Article  Google Scholar 

  29. 29

    Colin, E. et al. Akt is altered in an animal model of Huntington's disease and in patients. Eur. J. Neurosci. 21, 1478–1488 (2005).

    Article  Google Scholar 

  30. 30

    Berggard, T., Silow, M., Thulin, E. & Linse, S. Ca2+- and H+-dependent conformational changes of calbindin D28K . Biochemistry 39, 6864–6873 (2000).

    CAS  Article  Google Scholar 

  31. 31

    Cedervall, T. et al. Calbindin D28K EF-hand ligand binding and oligomerization: four high-affinity sites-three modes of action. Biochemistry 44, 13522–13532 (2005).

    CAS  Article  Google Scholar 

  32. 32

    Berggard, T. et al. Calbindin D28K exhibits properties characteristic of a Ca2+ sensor. J. Biol. Chem. 277, 16662–16672 (2002).

    CAS  Article  Google Scholar 

  33. 33

    Veenstra, T.D., Johnson, K.L., Tomlinson, A.J., Naylor, S. & Kumar, R. Determination of calcium-binding sites in rat brain calbindin D28K by electrospray ionization mass spectrometry. Biochemistry 36, 3535–3542 (1997).

    CAS  Article  Google Scholar 

  34. 34

    Vanbelle, C. et al. Deamidation and disulfide bridge formation in human calbindin D28K with effects on calcium binding. Protein Sci. 14, 968–979 (2005).

    CAS  Article  Google Scholar 

  35. 35

    Cedervall, T. et al. Redox sensitive cysteine residues in calbindin D28K are structurally and functionally important. Biochemistry 44, 684–693 (2005).

    CAS  Article  Google Scholar 

  36. 36

    Johnson, K.L. et al. On-line sample clean-up and chromatography coupled with electrospray ionization mass spectrometry to characterize the primary sequence and disulfide bond content of recombinant calcium binding proteins. Biomed. Chromatogr. 13, 37–45 (1999).

    CAS  Article  Google Scholar 

  37. 37

    Tao, L., Murphy, M.E. & English, A.M. S-nitrosation of Ca2+-loaded and Ca2+-free recombinant calbindin D28K from human brain. Biochemistry 41, 6185–6192 (2002).

    CAS  Article  Google Scholar 

  38. 38

    Nelson, M.R. & Chazin, W.J. Structures of EF-hand Ca2+-binding proteins: diversity in the organization, packing and response to Ca2+ binding. Biometals 11, 297–318 (1998).

    CAS  Article  Google Scholar 

  39. 39

    Klaus, W. et al. NMR investigation and secondary structure of domains I and II of rat brain calbindin D28K (1–93). Eur. J. Biochem. 262, 933–938 (1999).

    CAS  Article  Google Scholar 

  40. 40

    Mueller, G.A. et al. Global folds of proteins with low densities of NOEs using residual dipolar couplings: application to the 370-residue maltodextrin-binding protein. J. Mol. Biol. 300, 197–212 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Atreya, H.S. & Chary, K.V.R. New chemical shift signatures of bound calcium in EF-hand proteins. Curr. Sci. [online] 83, 1240–1245 (2002).

    CAS  Google Scholar 

  42. 42

    Biekofsky, R.R., Turjanski, A.G., Estrin, D.A., Feeney, J. & Pastore, A. Ab initio study of NMR 15N chemical shift differences induced by Ca2+ binding to EF-hand proteins. Biochemistry 43, 6554–6564 (2004).

    CAS  Article  Google Scholar 

  43. 43

    Linge, J.P., Williams, M.A., Spronk, C.A., Bonvin, A.M. & Nilges, M. Refinement of protein structures in explicit solvent. Proteins 50, 496–506 (2003).

    CAS  Article  Google Scholar 

  44. 44

    Rigden, D.J. & Galperin, M.Y. The DxDxDG motif for calcium binding: multiple structural contexts and implications for evolution. J. Mol. Biol. 343, 971–984 (2004).

    CAS  Article  Google Scholar 

  45. 45

    Akerfeldt, K.S., Coyne, A.N., Wilk, R.R., Thulin, E. & Linse, S. Ca2+-binding stoichiometry of calbindin D28K as assessed by spectroscopic analyses of synthetic peptide fragments. Biochemistry 35, 3662–3669 (1996).

    CAS  Article  Google Scholar 

  46. 46

    Donepudi, M. & Grutter, M.G. Structure and zymogen activation of caspases. Biophys. Chem. 101–102, 145–153 (2002).

    Article  Google Scholar 

  47. 47

    Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995).

    CAS  Article  Google Scholar 

  48. 48

    Johnson, B.A. & Blevins, R.A. NMRView—a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614 (1994).

    CAS  Article  Google Scholar 

  49. 49

    Helgstrand, M., Vanbelle, C., Thulin, E., Linse, S. & Akke, M. Sequential 1H, 15N and 13C NMR assignment of human calbindin D28K . J. Biomol. NMR 28, 305–306 (2004).

    CAS  Article  Google Scholar 

  50. 50

    Cornilescu, G., Delaglio, F. & Bax, A. Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302 (1999).

    CAS  Article  Google Scholar 

  51. 51

    Yang, D., Venters, R.A., Mueller, G.A., Choy, W.Y. & Kay, L.E. TROSY-based HNCO pulse sequences for the measurement of 1HN-15N, 15N-13CO, 1HN-13CO, 13CO-13Cα and 1HN-13Cα dipolar couplings in 15N, 13C, 2H-labeled proteins. J. Biomol. NMR 14, 333–343 (1999).

    Article  Google Scholar 

  52. 52

    Wang, Y.X. et al. Measurement of 3hJNC' connectivities across hydrogen bonds in a 30 kDa protein. J. Biomol. NMR 14, 181–184 (1999).

    CAS  Article  Google Scholar 

  53. 53

    Brunger, A.T. et al. Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921 (1998).

    CAS  Article  Google Scholar 

  54. 54

    Stein, E.G., Rice, L.M. & Brunger, A.T. Torsion-angle molecular dynamics as a new efficient tool for NMR structure calculation. J. Magn. Reson. 124, 154–164 (1997).

    CAS  Article  Google Scholar 

  55. 55

    Choy, W.Y., Tollinger, M., Mueller, G.A. & Kay, L.E. Direct structure refinement of high molecular weight proteins against residual dipolar couplings and carbonyl chemical shift changes upon alignment: an application to maltose binding protein. J. Biomol. NMR 21, 31–40 (2001).

    CAS  Article  Google Scholar 

  56. 56

    Nabuurs, S.B. et al. Quantitative evaluation of experimental NMR restraints. J. Am. Chem. Soc. 125, 12026–12034 (2003).

    CAS  Article  Google Scholar 

  57. 57

    Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. & Thornton, J.M. AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486 (1996).

    CAS  Article  Google Scholar 

  58. 58

    Davis, I.W., Murray, L.W., Richardson, J.S. & Richardson, D.C. MOLPROBITY: structure validation and all-atom contact analysis for nucleic acids and their complexes. Nucleic Acids Res. 32, W615–W619 (2004).

    CAS  Article  Google Scholar 

  59. 59

    Gouet, P., Robert, X. & Courcelle, E. ESPript/ENDscript: extracting and rendering sequence and 3D information from atomic structures of proteins. Nucleic Acids Res. 31, 3320–3323 (2003).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The Duke University NMR Center and the North Carolina State University NMR Facility were established with grants from the US National Institutes of Health, US National Science Foundation and North Carolina Biotechnology Center. We acknowledge support of this work by grants from the US National Institutes of Health (to R.K. and J.C.), the Kenan Institute for Engineering, Technology & Science (to J.C.) and the American Foundation for Aging Research (to D.J.K and D.R.K.).

Author information

Author notes

  1. Douglas J Kojetin and Ronald A Venters: These authors contributed equally to this work.

Affiliations

  1. Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, 27695, North Carolina, USA

    Douglas J Kojetin, David R Kordys, Richele J Thompson & John Cavanagh

  2. Duke University NMR Center, Duke University Medical Center, Durham, 27710, North Carolina, USA

    Ronald A Venters

  3. Departments of Biochemistry, Molecular Biology and Internal Medicine, Mayo Clinic and Foundation, Rochester, 55905, Minnesota, USA

    Rajiv Kumar

Authors
  1. Douglas J Kojetin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ronald A Venters
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. David R Kordys
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Richele J Thompson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Rajiv Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. John Cavanagh
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

D.J.K. contributed to assignments, structure calculations and manuscript preparation; R.A.V. to assignments, structure calculations, NMR data collection and analysis, and manuscript preparation; D.R.K. to sample preparations, fluorescence experiments and peptide-binding titrations; R.J.T. to sample preparations and NMR data collection; R.K. to RanBPM studies; and J.C. to NMR data collection and analysis, peptide-binding titrations and manuscript preparation.

Note: Supplementary information is available on the Nature Structural & Molecular Biology website.

Corresponding author

Correspondence to John Cavanagh.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Secondary structure and NOE statistics form Ca2+-loaded calbindin-D28K (PDF 5832 kb)

Supplementary Fig. 2

Measuring hydrophobic surface content via ANS fluorescence as calbindin-D28K binds Ca2+ (PDF 3759 kb)

Supplementary Table 1

NMR experiments used in the analysis of Ca2+-loaded calbindin-D28K (PDF 86 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Kojetin, D., Venters, R., Kordys, D. et al. Structure, binding interface and hydrophobic transitions of Ca2+-loaded calbindin-D28K. Nat Struct Mol Biol 13, 641–647 (2006). https://doi.org/10.1038/nsmb1112

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing