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Metal binding discrimination of the calmodulin Q41C/K75C mutant on Ca2+ and La3+

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Abstract

Calmodulin (CaM) is a multifunctional Ca2+-binding protein regulating the activity of many enzymes in response to fluctuation of the intracellular Ca2+ level. It has been shown that a CaM Q41C/K75C mutant (CaMSS) with a disulfide bond in the N-terminal domain exhibits greatly reduced affinity to Ca2+. In the present study, the experimental results revealed a unique metal binding pattern in CaMSS towards La3+ and Ca2+ separately: the mutant protein binds Ca2+ at site I, III and IV; however, it binds La3+ at site I, II and IV. A putative mechanism was proposed which is the conformation of site II (or site III) of CaMSS could be altered and thus loses its metal ion affinity in response to metal binding in the opposite terminal domain possibly through the long range domain interaction. The present work may offer new perspectives for understanding the mechanisms of specific metal ion affinity in CaM and for CaM-based protein design.

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References

  1. Zhang MJ, Toshiyuki T, Mitsuhiko I. Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nat Struct Biol, 1995, 2: 758–767

    Article  CAS  Google Scholar 

  2. Kuboniwa H, Tjandra N, Grzesiek S, Ren H, Klee CB, Bax A. Solution structure of calcium-free calmodulin. Nat Struct Biol, 1995, 2: 768–776

    Article  CAS  Google Scholar 

  3. Haiech J, Klee CB, Demaille JG, Haiech J. Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes. Theoretical approach to study of multiple ligand binding to a macromolecule. Biochemistry, 1981, 20: 3890–3897

    Article  CAS  Google Scholar 

  4. Wang C-LA, Aquaron RR, Leavis PC, Gergely J. Metal-binding properties of calmodulin. Eur J Biochem, 1982, 124: 7–12

    Article  CAS  Google Scholar 

  5. Ye Y, Lee HW, Yang W, Shealy S, Yang JJ. Probing site-specific calmodulin calcium and lanthanide affinity by grafting. J Am Chem Soc, 2005, 127: 3743–3750

    Article  CAS  Google Scholar 

  6. Maune JF, Beckingham K, Martin SR, Bayley PM. Circular dichroism studies on calcium binding to two series of calcium binding site mutants of drosophila melanogaster calmodulin. Biochemistry, 1992, 31: 7779–7786

    Article  CAS  Google Scholar 

  7. Martin SR, Maune JF, Beckingham K, Bayley PM. Stopped-flow studies of calcium dissociation for calcium-binding-site mutants of drosophila melanogaster calmodulin. Eur J Biochem, 1992, 205: 1107–1114

    Article  CAS  Google Scholar 

  8. Starovasnik M, Su DR, Beckingham K, Klevit RE. A series of point mutations reveal interactions between the calcium-binding sites of calmodulin. Protein Sci, 1992, 1: 245–253

    Article  CAS  Google Scholar 

  9. Yang Q, Hu J, Yang X, Wang K. Mastoparan/mastoparan x altered binding behavior of La3+ to calmodulin in ternary complexes. J Inorg Biochem, 2008, 102: 278–284

    Article  CAS  Google Scholar 

  10. Tan RY, Mabuchi Y, Grabarek Z. Blocking the Ca2+-induced conformational transitions in calmodulin with disulfide bonds. J Biol Chem, 1996, 271: 7479–7483

    Article  CAS  Google Scholar 

  11. Grabarek Z. Structure of a trapped intermediate of calmodulin: calcium regulation of EF-hand proteins from a new perspective. J Mol Biol, 2005, 346: 1351–1366

    Article  CAS  Google Scholar 

  12. Hu J, Jia X, Li Q, Yang X, Wang K. Binding of La3+ to calmodulin and its effects on the interaction between calmodulin and calmodulin binding peptide, polistes mastoparan. Biochemistry, 2004, 43: 2688–2698

    Article  CAS  Google Scholar 

  13. Bowers WF, Fluton S, Thompson J. Clin Pharmacokinet, 1984, 9: 49–60

    Article  CAS  Google Scholar 

  14. Chang W, Li ka. Concise Handbook of Analytical Chemistry. Beijing: Peking University publishing company, 1981

    Google Scholar 

  15. Sun YS. The Physical and Chemical Constants of Rare Earth Elements. Beijing: Metallurgy Press, 1978

    Google Scholar 

  16. Xu K, Yang XD, Wang K. La3+ induced binding of calmodulin (CaM) to CaM-binding proteins in rat brain homogenate. Chem J Chin Univ, 2008, 29: 2525–2530

    CAS  Google Scholar 

  17. Tiandra N, Kuboniwa H, Ren H, Bax A. Rotational dynamics of calcium-free calmodulin studied by NMR relaxation measurements. Eur Jof Biochem, 1995, 230: 1014–1024

    Article  Google Scholar 

  18. Urbauer JL, Short JH, Dow LK, Wand AJ. Structural analysis of a novel interaction by calmodulin: high-affinity binding of a peptide in the absence of calcium. Biochemistry, 1995, 34: 8099–8109

    Article  CAS  Google Scholar 

  19. Mitsuhiko I, Marion D, Kay LE, Shih H, Krinks M, Klee CB, Bax A. Heteronuclear 3D NMR and isotopic labeling of calmodulin: towards the complete assignment of the 1h NMR spectrum. Biochem Pharm, 1990, 40: 153–160

    Article  Google Scholar 

  20. Wendy SV, Brenda RS, Elena R, William RL. Calcium binding to calmodulin mutants monitored by domain-specific intrinsic phenylalanine and tyrosine fluorescence. Biophys J, 2002, 83: 2767

    Article  Google Scholar 

  21. Ohki Sy, Ikura M, Zhang M. Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin. Biochemistry, 1997, 36: 4309–4316

    Article  CAS  Google Scholar 

  22. Ouyang H, Vogel HJ. Metal ion binding to calmodulin: NMR and fluorescence studies. BioMetals, 1998, 11: 213–222

    Article  CAS  Google Scholar 

  23. Dean JA. Lang’s handbook of chemistry, 2nd ed. Beijing: Science Press, 2003

    Google Scholar 

  24. Elad P, Ran F, Esther N, Menachem G. A molecular dynamics study of the effect of Ca2+ removal on calmodulin structure. Biophys J, 2006, 90: 3842

    Article  CAS  Google Scholar 

  25. Ababou A, Shenvi RA, Desjarlais JR. Long-range effects on calcium binding and conformational change in the N-domain of calmodulin. Biochemistry, 2001, 40: 12719–12726

    Article  CAS  Google Scholar 

  26. Ikura M, Hasegawa N, Aimoto S, Yazawa M, Yagi K, Hikichi K. 113Cd-NMR evidence for cooperative interaction between amino- and carboxyl-terminal domains of calmodulin. Biochem Biophys Res Commun, 1989, 161: 1233–1238

    Article  CAS  Google Scholar 

  27. Pedigo S, Shea MA. Quantitative endoproteinase gluc footprinting of cooperative Ca2+ binding to calmodulin: Proteolytic susceptibility of E31 and E87 indicates interdomain interactions. Biochemistry, 1995, 34: 1179–1196

    Article  CAS  Google Scholar 

  28. Medvedeva MV, Polyakova OV, Watterson DM, Gusev NB. Mutation of Lys-75 affects calmodulin conformation. FEBS Letters, 1999, 450: 139–143

    Article  CAS  Google Scholar 

  29. Nelson MR, Chazin WJ. Structures of EF-hand Ca2+-binding proteins: diversity in the organization, packing and response to Ca2+ binding. BioMetals, 1998, 11: 297–318

    Article  CAS  Google Scholar 

  30. Grabarek Z. Structural basis for diversity of the EF-hand calcium-binding proteins. J Mol Biol, 2006, 359: 509–525

    Article  CAS  Google Scholar 

  31. Yiming Y, Hsiau-Wei L, Wei Y, Sarah JS, Anna LW, Zhi-ren L, Ivan T, Robert H, Robert W, Jenny JY. Metal binding affinity and structural properties of an isolated EF-loop in a scaffold protein. Protein Eng, 2001, 14: 1001

    Article  Google Scholar 

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Authors and Affiliations

  1. State Key Laboratory for Natural and Biomimetic Drugs, Peking University Health Science Center, Beijing, 100191, China

    Kun Xu, XiaoDa Yang & Kui Wang

  2. Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University Health Science Center, Beijing, 100191, China

    Kun Xu, XiaoDa Yang & Kui Wang

Authors
  1. Kun Xu
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  2. XiaoDa Yang
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  3. Kui Wang
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Correspondence to XiaoDa Yang.

Additional information

Support from the National Natural Science Foundation of China (Grant Nos. 20671008 & 20637010) and the Key Construction Program of the National “985” Project. Dr. Changwen Jin, Dr. Xianrong Guo and Dr. You Li at Beijing NMR Center, Peking University, are gratefully acknowledged for their assistance in data collection and analysis of Bruker DRX 600 NMR spectrometer. We also thank Dr. T. Squier at University of Kansas for the generous gift of the plasmid encoding gene of chicken calmodulin.

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Xu, K., Yang, X. & Wang, K. Metal binding discrimination of the calmodulin Q41C/K75C mutant on Ca2+ and La3+ . Sci. China Chem. 53, 797–806 (2010). https://doi.org/10.1007/s11426-010-0059-2

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  • DOI: https://doi.org/10.1007/s11426-010-0059-2

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