Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2000 May;9(5):964–975. doi: 10.1110/ps.9.5.964

Peptide and metal ion-dependent association of isolated helix-loop-helix calcium binding domains: studies of thrombic fragments of calmodulin.

R D Brokx 1, H J Vogel 1
PMCID: PMC2144632  PMID: 10850806

Abstract

Calmodulin (CaM), the ubiquitous, eukaryotic, bilobal calcium-binding regulatory protein, has been cleaved by thrombin to create two fragments. TM1 (1-106) and TM2 (107-148). NMR and CD results indicate that TMI and TM2 can associate in the presence of Ca2+ to form a complex similar to native CaM, even though the cleavage site is not in the linker region between two helix-loop-helix domains, but rather within an alpha-helix. Cadmium-113 NMR results show that this complex has enhanced metal-ion binding properties when compared to either TM1 or TM2 alone. This complex can bind several CaM-binding target peptides, as shown by gel bandshift assays, circular dichroism spectra, and 13C NMR spectra of biosynthetically methyl-13C-Met-labeled TM1 and TM2; moreover, gel bandshift assays show that the addition of a target peptide strengthens the interactions between TM1 and TM2 and increases the stability of the complex. Cadmium-113 NMR spectra indicate that the TM1:TM2 complex can also bind the antipsychotic drug trifluoperazine. However, in contrast to CaM:peptide complexes, the TM1:TM2:peptide complexes are disrupted by 4 M urea; moreover, TM1 and TM2 in combination are unable to activate CaM-dependent enzymes. This suggests that TM1:TM2 mixtures cannot bind target molecules as tightly as intact CaM, or perhaps that binding occurs but additional interactions with the target enzymes that are necessary for proper activation are perturbed by the proteolytic cleavage. The results presented here reflect the importance of the existence of helix-loop-helix Ca2+-binding domains in pairs in proteins such as CaM, and extend the understanding of the association of such domains in this class of proteins in general.

Full Text

The Full Text of this article is available as a PDF (524.9 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akke M., Forsén S., Chazin W. J. Molecular basis for co-operativity in Ca2+ binding to calbindin D9k. 1H nuclear magnetic resonance studies of (Cd2+)1-bovine calbindin D9k. J Mol Biol. 1991 Jul 5;220(1):173–189. doi: 10.1016/0022-2836(91)90389-n. [DOI] [PubMed] [Google Scholar]
  2. Akke M., Forsén S., Chazin W. J. Solution structure of (Cd2+)1-calbindin D9k reveals details of the stepwise structural changes along the Apo-->(Ca2+)II1-->(Ca2+)I,II2 binding pathway. J Mol Biol. 1995 Sep 8;252(1):102–121. doi: 10.1006/jmbi.1995.0478. [DOI] [PubMed] [Google Scholar]
  3. Andersson A., Forsén S., Thulin E., Vogel H. J. Cadmium-113 nuclear magnetic resonance studies of proteolytic fragments of calmodulin: assignment of strong and weak cation binding sites. Biochemistry. 1983 May 10;22(10):2309–2313. doi: 10.1021/bi00279a001. [DOI] [PubMed] [Google Scholar]
  4. Aramini J. M., Hiraoki T., Ke Y., Nitta K., Vogel H. J. Cadmium-113 NMR studies of bovine and human alpha-lactalbumin and equine lysozyme. J Biochem. 1995 Mar;117(3):623–628. doi: 10.1093/oxfordjournals.jbchem.a124754. [DOI] [PubMed] [Google Scholar]
  5. Babu Y. S., Bugg C. E., Cook W. J. Structure of calmodulin refined at 2.2 A resolution. J Mol Biol. 1988 Nov 5;204(1):191–204. doi: 10.1016/0022-2836(88)90608-0. [DOI] [PubMed] [Google Scholar]
  6. Barbato G., Ikura M., Kay L. E., Pastor R. W., Bax A. Backbone dynamics of calmodulin studied by 15N relaxation using inverse detected two-dimensional NMR spectroscopy: the central helix is flexible. Biochemistry. 1992 Jun 16;31(23):5269–5278. doi: 10.1021/bi00138a005. [DOI] [PubMed] [Google Scholar]
  7. Barth A., Martin S. R., Bayley P. M. Resolution of Trp near UV CD spectra of calmodulin-domain peptide complexes into the 1La and 1Lb component spectra. Biopolymers. 1998 Jun;45(7):493–501. doi: 10.1002/(SICI)1097-0282(199806)45:7<493::AID-BIP3>3.0.CO;2-J. [DOI] [PubMed] [Google Scholar]
  8. Caday C. G., Steiner R. F. The interaction of calmodulin with melittin. Biochem Biophys Res Commun. 1986 Mar 13;135(2):419–425. doi: 10.1016/0006-291x(86)90011-2. [DOI] [PubMed] [Google Scholar]
  9. Chattopadhyaya R., Meador W. E., Means A. R., Quiocho F. A. Calmodulin structure refined at 1.7 A resolution. J Mol Biol. 1992 Dec 20;228(4):1177–1192. doi: 10.1016/0022-2836(92)90324-d. [DOI] [PubMed] [Google Scholar]
  10. Delaglio F., Grzesiek S., Vuister G. W., Zhu G., Pfeifer J., Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995 Nov;6(3):277–293. doi: 10.1007/BF00197809. [DOI] [PubMed] [Google Scholar]
  11. Delaglio F., Grzesiek S., Vuister G. W., Zhu G., Pfeifer J., Bax A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR. 1995 Nov;6(3):277–293. doi: 10.1007/BF00197809. [DOI] [PubMed] [Google Scholar]
  12. Erickson-Viitanen S., DeGrado W. F. Recognition and characterization of calmodulin-binding sequences in peptides and proteins. Methods Enzymol. 1987;139:455–478. doi: 10.1016/0076-6879(87)39106-2. [DOI] [PubMed] [Google Scholar]
  13. Finn B. E., Kördel J., Thulin E., Sellers P., Forsén S. Dissection of calbindin D9k into two Ca(2+)-binding subdomains by a combination of mutagenesis and chemical cleavage. FEBS Lett. 1992 Feb 24;298(2-3):211–214. doi: 10.1016/0014-5793(92)80059-p. [DOI] [PubMed] [Google Scholar]
  14. Fontana A., Fassina G., Vita C., Dalzoppo D., Zamai M., Zambonin M. Correlation between sites of limited proteolysis and segmental mobility in thermolysin. Biochemistry. 1986 Apr 22;25(8):1847–1851. doi: 10.1021/bi00356a001. [DOI] [PubMed] [Google Scholar]
  15. Forsén S., Thulin E., Drakenberg T., Krebs J., Seamon K. A 113Cd NMR study of calmodulin and its interaction with calcium, magnesium and trifluoperazine. FEBS Lett. 1980 Aug 11;117(1):189–194. doi: 10.1016/0014-5793(80)80942-2. [DOI] [PubMed] [Google Scholar]
  16. Gagné S. M., Tsuda S., Li M. X., Chandra M., Smillie L. B., Sykes B. D. Quantification of the calcium-induced secondary structural changes in the regulatory domain of troponin-C. Protein Sci. 1994 Nov;3(11):1961–1974. doi: 10.1002/pro.5560031108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gariépy J., Sykes B. D., Reid R. E., Hodges R. S. Proton nuclear magnetic resonance investigation of synthetic calcium-binding peptides. Biochemistry. 1982 Mar 30;21(7):1506–1512. doi: 10.1021/bi00536a007. [DOI] [PubMed] [Google Scholar]
  18. Gerendasy D. D., Herron S. R., Jennings P. A., Sutcliffe J. G. Calmodulin stabilizes an amphiphilic alpha-helix within RC3/neurogranin and GAP-43/neuromodulin only when Ca2+ is absent. J Biol Chem. 1995 Mar 24;270(12):6741–6750. doi: 10.1074/jbc.270.12.6741. [DOI] [PubMed] [Google Scholar]
  19. Gopalakrishna R., Anderson W. B. Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography. Biochem Biophys Res Commun. 1982 Jan 29;104(2):830–836. doi: 10.1016/0006-291x(82)90712-4. [DOI] [PubMed] [Google Scholar]
  20. Graziano G., Catanzano F., Giancola C., Barone G. DSC study of the thermal stability of S-protein and S-peptide/S-protein. Biochemistry. 1996 Oct 15;35(41):13386–13392. doi: 10.1021/bi960856+. [DOI] [PubMed] [Google Scholar]
  21. Hubbard S. J. The structural aspects of limited proteolysis of native proteins. Biochim Biophys Acta. 1998 Feb 17;1382(2):191–206. doi: 10.1016/s0167-4838(97)00175-1. [DOI] [PubMed] [Google Scholar]
  22. Ikura M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci. 1996 Jan;21(1):14–17. [PubMed] [Google Scholar]
  23. Ikura M., Clore G. M., Gronenborn A. M., Zhu G., Klee C. B., Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science. 1992 May 1;256(5057):632–638. doi: 10.1126/science.1585175. [DOI] [PubMed] [Google Scholar]
  24. Ikura M., Hiraoki T., Hikichi K., Mikuni T., Yazawa M., Yagi K. Nuclear magnetic resonance studies on calmodulin: calcium-induced conformational change. Biochemistry. 1983 May 10;22(10):2573–2579. doi: 10.1021/bi00279a039. [DOI] [PubMed] [Google Scholar]
  25. Ikura M., Kay L. E., Bax A. A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. Biochemistry. 1990 May 15;29(19):4659–4667. doi: 10.1021/bi00471a022. [DOI] [PubMed] [Google Scholar]
  26. Ikura M., Spera S., Barbato G., Kay L. E., Krinks M., Bax A. Secondary structure and side-chain 1H and 13C resonance assignments of calmodulin in solution by heteronuclear multidimensional NMR spectroscopy. Biochemistry. 1991 Sep 24;30(38):9216–9228. doi: 10.1021/bi00102a013. [DOI] [PubMed] [Google Scholar]
  27. Juminaga D., Albaugh S. A., Steiner R. F. The interaction of calmodulin with regulatory peptides of phosphorylase kinase. J Biol Chem. 1994 Jan 21;269(3):1660–1667. [PubMed] [Google Scholar]
  28. Kay L. E., Forman-Kay J. D., McCubbin W. D., Kay C. M. Solution structure of a polypeptide dimer comprising the fourth Ca(2+)-binding site of troponin C by nuclear magnetic resonance spectroscopy. Biochemistry. 1991 Apr 30;30(17):4323–4333. doi: 10.1021/bi00231a031. [DOI] [PubMed] [Google Scholar]
  29. Kippen A. D., Sancho J., Fersht A. R. Folding of barnase in parts. Biochemistry. 1994 Mar 29;33(12):3778–3786. doi: 10.1021/bi00178a039. [DOI] [PubMed] [Google Scholar]
  30. Kuboniwa H., Tjandra N., Grzesiek S., Ren H., Klee C. B., Bax A. Solution structure of calcium-free calmodulin. Nat Struct Biol. 1995 Sep;2(9):768–776. doi: 10.1038/nsb0995-768. [DOI] [PubMed] [Google Scholar]
  31. Linse S., Helmersson A., Forsén S. Calcium binding to calmodulin and its globular domains. J Biol Chem. 1991 May 5;266(13):8050–8054. [PubMed] [Google Scholar]
  32. Linse S., Thulin E., Gifford L. K., Radzewsky D., Hagan J., Wilk R. R., Akerfeldt K. S. Domain organization of calbindin D28k as determined from the association of six synthetic EF-hand fragments. Protein Sci. 1997 Nov;6(11):2385–2396. doi: 10.1002/pro.5560061112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Linse S., Thulin E., Sellers P. Disulfide bonds in homo- and heterodimers of EF-hand subdomains of calbindin D9k: stability, calcium binding, and NMR studies. Protein Sci. 1993 Jun;2(6):985–1000. doi: 10.1002/pro.5560020612. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  35. Martin S. R., Bayley P. M. The effects of Ca2+ and Cd2+ on the secondary and tertiary structure of bovine testis calmodulin. A circular-dichroism study. Biochem J. 1986 Sep 1;238(2):485–490. doi: 10.1042/bj2380485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Meador W. E., Means A. R., Quiocho F. A. Modulation of calmodulin plasticity in molecular recognition on the basis of x-ray structures. Science. 1993 Dec 10;262(5140):1718–1721. doi: 10.1126/science.8259515. [DOI] [PubMed] [Google Scholar]
  37. Meador W. E., Means A. R., Quiocho F. A. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science. 1992 Aug 28;257(5074):1251–1255. doi: 10.1126/science.1519061. [DOI] [PubMed] [Google Scholar]
  38. Means A. R., VanBerkum M. F., Bagchi I., Lu K. P., Rasmussen C. D. Regulatory functions of calmodulin. Pharmacol Ther. 1991;50(2):255–270. doi: 10.1016/0163-7258(91)90017-g. [DOI] [PubMed] [Google Scholar]
  39. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Newton D. L., Oldewurtel M. D., Krinks M. H., Shiloach J., Klee C. B. Agonist and antagonist properties of calmodulin fragments. J Biol Chem. 1984 Apr 10;259(7):4419–4426. [PubMed] [Google Scholar]
  41. O'Neil K. T., DeGrado W. F. How calmodulin binds its targets: sequence independent recognition of amphiphilic alpha-helices. Trends Biochem Sci. 1990 Feb;15(2):59–64. doi: 10.1016/0968-0004(90)90177-d. [DOI] [PubMed] [Google Scholar]
  42. Ouyang H., Vogel H. J. Melatonin and serotonin interactions with calmodulin: NMR, spectroscopic and biochemical studies. Biochim Biophys Acta. 1998 Mar 3;1383(1):37–47. doi: 10.1016/s0167-4838(97)00157-x. [DOI] [PubMed] [Google Scholar]
  43. Palomo-Jiménez P. I., Hernández-Hernando S., García-Nieto R. M., Villalobo A. A method for the purification of phospho(Tyr)calmodulin free of nonphosphorylated calmodulin. Protein Expr Purif. 1999 Aug;16(3):388–395. doi: 10.1006/prep.1999.1092. [DOI] [PubMed] [Google Scholar]
  44. Putkey J. A., Slaughter G. R., Means A. R. Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene. J Biol Chem. 1985 Apr 25;260(8):4704–4712. [PubMed] [Google Scholar]
  45. Reid R. E., Gariépy J., Saund A. K., Hodges R. S. Calcium-induced protein folding. Structure-affinity relationships in synthetic analogs of the helix-loop-helix calcium binding unit. J Biol Chem. 1981 Mar 25;256(6):2742–2751. [PubMed] [Google Scholar]
  46. Shaw G. S., Hodges R. S., Sykes B. D. Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence. Science. 1990 Jul 20;249(4966):280–283. doi: 10.1126/science.2374927. [DOI] [PubMed] [Google Scholar]
  47. Shaw G. S., Sykes B. D. NMR solution structure of a synthetic troponin C heterodimeric domain. Biochemistry. 1996 Jun 11;35(23):7429–7438. doi: 10.1021/bi9528006. [DOI] [PubMed] [Google Scholar]
  48. Shea M. A., Verhoeven A. S., Pedigo S. Calcium-induced interactions of calmodulin domains revealed by quantitative thrombin footprinting of Arg37 and Arg106. Biochemistry. 1996 Mar 5;35(9):2943–2957. doi: 10.1021/bi951934g. [DOI] [PubMed] [Google Scholar]
  49. Strynadka N. C., James M. N. Crystal structures of the helix-loop-helix calcium-binding proteins. Annu Rev Biochem. 1989;58:951–998. doi: 10.1146/annurev.bi.58.070189.004511. [DOI] [PubMed] [Google Scholar]
  50. Su Z., Fan D., George S. E. Role of domain 3 of calmodulin in activation of calmodulin-stimulated phosphodiesterase and smooth muscle myosin light chain kinase. J Biol Chem. 1994 Jun 17;269(24):16761–16765. [PubMed] [Google Scholar]
  51. Sudmeier J. L., Bell S. J., Storm M. C., Dunn M. F. Cadmium-113 nuclear magnetic resonance studies of bovine insulin: two-zinc insulin hexamer specifically binds calcium. Science. 1981 May 1;212(4494):560–562. doi: 10.1126/science.7010607. [DOI] [PubMed] [Google Scholar]
  52. Tasayco M. L., Chao K. NMR study of the reconstitution of the beta-sheet of thioredoxin by fragment complementation. Proteins. 1995 May;22(1):41–44. doi: 10.1002/prot.340220106. [DOI] [PubMed] [Google Scholar]
  53. Thulin E., Andersson A., Drakenberg T., Forsén S., Vogel H. J. Metal ion and drug binding to proteolytic fragments of calmodulin: proteolytic, cadmium-113, and proton nuclear magnetic resonance studies. Biochemistry. 1984 Apr 10;23(8):1862–1870. doi: 10.1021/bi00303a043. [DOI] [PubMed] [Google Scholar]
  54. Vogel H. J. The Merck Frosst Award Lecture 1994. Calmodulin: a versatile calcium mediator protein. Biochem Cell Biol. 1994 Sep-Oct;72(9-10):357–376. [PubMed] [Google Scholar]
  55. Vogel H. J., Zhang M. Protein engineering and NMR studies of calmodulin. Mol Cell Biochem. 1995 Aug-Sep;149-150:3–15. doi: 10.1007/BF01076558. [DOI] [PubMed] [Google Scholar]
  56. Wall C. M., Grand R. J., Perry S. V. Biological activities of the peptides obtained by digestion of troponin C and calmodulin with thrombin. Biochem J. 1981 Apr 1;195(1):307–316. doi: 10.1042/bj1950307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Walsh M., Stevens F. C. Characterization of tryptic fragments obtained from bovine brain protein modulator of cyclic nucleotide phosphodiesterase. J Biol Chem. 1977 Nov 10;252(21):7440–7443. [PubMed] [Google Scholar]
  58. Waltersson Y., Linse S., Brodin P., Grundström T. Mutational effects on the cooperativity of Ca2+ binding in calmodulin. Biochemistry. 1993 Aug 10;32(31):7866–7871. doi: 10.1021/bi00082a005. [DOI] [PubMed] [Google Scholar]
  59. Wang J. H., Teo T. S., Wang T. H. Hysteretic substrate activation of bovine heart c-AMP phosphodiestrase. Biochem Biophys Res Commun. 1972 Feb 16;46(3):1306–1311. doi: 10.1016/s0006-291x(72)80117-7. [DOI] [PubMed] [Google Scholar]
  60. Weber L. P., Van Lierop J. E., Walsh M. P. Ca2+-independent phosphorylation of myosin in rat caudal artery and chicken gizzard myofilaments. J Physiol. 1999 May 1;516(Pt 3):805–824. doi: 10.1111/j.1469-7793.1999.0805u.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wójcik J., Góral J., Pawłowski K., Bierzyński A. Isolated calcium-binding loops of EF-hand proteins can dimerize to form a native-like structure. Biochemistry. 1997 Jan 28;36(4):680–687. doi: 10.1021/bi961821c. [DOI] [PubMed] [Google Scholar]
  62. Yuan T., Mietzner T. A., Montelaro R. C., Vogel H. J. Characterization of the calmodulin binding domain of SIV transmembrane glycoprotein by NMR and CD spectroscopy. Biochemistry. 1995 Aug 22;34(33):10690–10696. doi: 10.1021/bi00033a045. [DOI] [PubMed] [Google Scholar]
  63. Yuan T., Ouyang H., Vogel H. J. Surface exposure of the methionine side chains of calmodulin in solution. A nitroxide spin label and two-dimensional NMR study. J Biol Chem. 1999 Mar 26;274(13):8411–8420. doi: 10.1074/jbc.274.13.8411. [DOI] [PubMed] [Google Scholar]
  64. Yuan T., Vogel H. J., Sutherland C., Walsh M. P. Characterization of the Ca2+ -dependent and -independent interactions between calmodulin and its binding domain of inducible nitric oxide synthase. FEBS Lett. 1998 Jul 17;431(2):210–214. doi: 10.1016/s0014-5793(98)00750-9. [DOI] [PubMed] [Google Scholar]
  65. Yuan T., Walsh M. P., Sutherland C., Fabian H., Vogel H. J. Calcium-dependent and -independent interactions of the calmodulin-binding domain of cyclic nucleotide phosphodiesterase with calmodulin. Biochemistry. 1999 Feb 2;38(5):1446–1455. doi: 10.1021/bi9816453. [DOI] [PubMed] [Google Scholar]
  66. Yuan T., Weljie A. M., Vogel H. J. Tryptophan fluorescence quenching by methionine and selenomethionine residues of calmodulin: orientation of peptide and protein binding. Biochemistry. 1998 Mar 3;37(9):3187–3195. doi: 10.1021/bi9716579. [DOI] [PubMed] [Google Scholar]
  67. Zhang M., Fabian H., Mantsch H. H., Vogel H. J. Isotope-edited Fourier transform infrared spectroscopy studies of calmodulin's interaction with its target peptides. Biochemistry. 1994 Sep 13;33(36):10883–10888. doi: 10.1021/bi00202a006. [DOI] [PubMed] [Google Scholar]
  68. Zhang M., Tanaka T., Ikura M. Calcium-induced conformational transition revealed by the solution structure of apo calmodulin. Nat Struct Biol. 1995 Sep;2(9):758–767. doi: 10.1038/nsb0995-758. [DOI] [PubMed] [Google Scholar]
  69. Zhang M., Vogel H. J. Determination of the side chain pKa values of the lysine residues in calmodulin. J Biol Chem. 1993 Oct 25;268(30):22420–22428. [PubMed] [Google Scholar]
  70. Zhang M., Vogel H. J., Zwiers H. Nuclear magnetic resonance studies of the structure of B50/neuromodulin and its interaction with calmodulin. Biochem Cell Biol. 1994 Mar-Apr;72(3-4):109–116. doi: 10.1139/o94-017. [DOI] [PubMed] [Google Scholar]
  71. de Prat Gay G., Fersht A. R. Generation of a family of protein fragments for structure-folding studies. 1. Folding complementation of two fragments of chymotrypsin inhibitor-2 formed by cleavage at its unique methionine residue. Biochemistry. 1994 Jun 28;33(25):7957–7963. doi: 10.1021/bi00191a024. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES