Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 1999 Jun 1;340(Pt 2):417–424.

Structural elements within the methylation loop (residues 112-117) and EF hands III and IV of calmodulin are required for Lys(115) trimethylation.

J A Cobb 1, C H Han 1, D M Wills 1, D M Roberts 1
PMCID: PMC1220266  PMID: 10333484

Abstract

Calmodulin is trimethylated by a specific methyltransferase on Lys115, a residue located in a six amino acid loop (LGEKLT) between EF hands III and IV. To investigate the structural requirements for methylation, domain exchange mutants as well as single point mutations of conserved methylation loop residues (E114A, Glu114-->Ala; L116T, Leu116-->Thr) were generated. E114A and L116T activated cyclic nucleotide phosphodiesterase (PDE) and NAD+ kinase (NADK) similar to wild-type calmodulin, but lost their ability to be methylated. Domain exchange mutants in which EF hand III or IV was replaced by EF hand I or II respectively (CaM1214 and CaM1232 respectively) showed a modest effect on PDE and NADK activation (50 to 100% of wild-type), but calmodulin methylation was abolished. A third domain exchange mutant, CaMEKL, has the methylation loop sequence placed at a symmetrical position between EF hands I and II in the N-terminal lobe [residues QNP(41-43) replaced by EKL]. CaMEKL activated PDE normally, but did not activate NADK. However, CaMEKL retained the ability to bind to NADK and inhibited activation by wild-type calmodulin. Site-directed mutagenesis of single residues showed that Gln41 and Pro43 substitutions had the strongest effect on NADK activation. Additionally, CaMEKL was not methylated, suggesting that the introduction of the methylation loop between EF hands I and II is not adequate for methyltransferase recognition. Overall the data indicate that residues in the methylation loop are essential but not sufficient for methyltransferase recognition, and that additional residues unique to EF hands III and IV are required. Secondly, the QNP sequence in the loop between EF hands I and II is necessary for NADK activation.

Full Text

The Full Text of this article is available as a PDF (195.8 KB).

Selected References

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

  1. 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]
  2. 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]
  3. Blumenthal D. K., Stull J. T. Effects of pH, ionic strength, and temperature on activation by calmodulin an catalytic activity of myosin light chain kinase. Biochemistry. 1982 May 11;21(10):2386–2391. doi: 10.1021/bi00539a017. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Crouch T. H., Klee C. B. Positive cooperative binding of calcium to bovine brain calmodulin. Biochemistry. 1980 Aug 5;19(16):3692–3698. doi: 10.1021/bi00557a009. [DOI] [PubMed] [Google Scholar]
  6. George S. E., Su Z., Fan D., Means A. R. Calmodulin-cardiac troponin C chimeras. Effects of domain exchange on calcium binding and enzyme activation. J Biol Chem. 1993 Nov 25;268(33):25213–25220. [PubMed] [Google Scholar]
  7. George S. E., VanBerkum M. F., Ono T., Cook R., Hanley R. M., Putkey J. A., Means A. R. Chimeric calmodulin-cardiac troponin C proteins differentially activate calmodulin target enzymes. J Biol Chem. 1990 Jun 5;265(16):9228–9235. [PubMed] [Google Scholar]
  8. Gregori L., Marriott D., West C. M., Chau V. Specific recognition of calmodulin from Dictyostelium discoideum by the ATP, ubiquitin-dependent degradative pathway. J Biol Chem. 1985 May 10;260(9):5232–5235. [PubMed] [Google Scholar]
  9. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  10. Han C. H., Richardson J., Oh S. H., Roberts D. M. Isolation and kinetic characterization of the calmodulin methyltransferase from sheep brain. Biochemistry. 1993 Dec 21;32(50):13974–13980. doi: 10.1021/bi00213a030. [DOI] [PubMed] [Google Scholar]
  11. Han C. H., Roberts D. M. Altered methylation substrate kinetics and calcium binding of a calmodulin with a Val136-->Thr substitution. Eur J Biochem. 1997 Mar 15;244(3):904–912. doi: 10.1111/j.1432-1033.1997.00904.x. [DOI] [PubMed] [Google Scholar]
  12. Harding S. A., Oh S. H., Roberts D. M. Transgenic tobacco expressing a foreign calmodulin gene shows an enhanced production of active oxygen species. EMBO J. 1997 Mar 17;16(6):1137–1144. doi: 10.1093/emboj/16.6.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ikura M. Calcium binding and conformational response in EF-hand proteins. Trends Biochem Sci. 1996 Jan;21(1):14–17. [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. Lee S. H., Seo H. Y., Kim J. C., Heo W. D., Chung W. S., Lee K. J., Kim M. C., Cheong Y. H., Choi J. Y., Lim C. O. Differential activation of NAD kinase by plant calmodulin isoforms. The critical role of domain I. J Biol Chem. 1997 Apr 4;272(14):9252–9259. doi: 10.1074/jbc.272.14.9252. [DOI] [PubMed] [Google Scholar]
  17. Lukas T. J., Wallen-Friedman M., Kung C., Watterson D. M. In vivo mutations of calmodulin: a mutant Paramecium with altered ion current regulation has an isoleucine-to-threonine change at residue 136 and an altered methylation state at lysine residue 115. Proc Natl Acad Sci U S A. 1989 Oct;86(19):7331–7335. doi: 10.1073/pnas.86.19.7331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Morino H., Kawamoto T., Miyake M., Kakimoto Y. Purification and properties of calmodulin-lysine N-methyltransferase from rat brain cytosol. J Neurochem. 1987 Apr;48(4):1201–1208. doi: 10.1111/j.1471-4159.1987.tb05647.x. [DOI] [PubMed] [Google Scholar]
  20. Nelson H. B., Heiman R. G., Bolduc C., Kovalick G. E., Whitley P., Stern M., Beckingham K. Calmodulin point mutations affect Drosophila development and behavior. Genetics. 1997 Dec;147(4):1783–1798. doi: 10.1093/genetics/147.4.1783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Newton D. L., Burke T. R., Jr, Rice K. C., Klee C. B. Calcium ion dependent covalent modification of calmodulin with norchlorpromazine isothiocyanate. Biochemistry. 1983 Nov 22;22(24):5472–5476. doi: 10.1021/bi00293a003. [DOI] [PubMed] [Google Scholar]
  22. Oh S. H., Roberts D. M. Analysis of the state of posttranslational calmodulin methylation in developing pea plants. Plant Physiol. 1990 Jul;93(3):880–887. doi: 10.1104/pp.93.3.880. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pech L. L., Nelson D. L. Purification and characterization of calmodulin (lysine 115) N-methyltransferase from Paramecium tetraurelia. Biochim Biophys Acta. 1994 Mar 2;1199(2):183–194. doi: 10.1016/0304-4165(94)90114-7. [DOI] [PubMed] [Google Scholar]
  24. Persechini A., Gansz K. J., Paresi R. J. Activation of myosin light chain kinase and nitric oxide synthase activities by engineered calmodulins with duplicated or exchanged EF hand pairs. Biochemistry. 1996 Jan 9;35(1):224–228. doi: 10.1021/bi952383x. [DOI] [PubMed] [Google Scholar]
  25. Persechini A., Kretsinger R. H. The central helix of calmodulin functions as a flexible tether. J Biol Chem. 1988 Sep 5;263(25):12175–12178. [PubMed] [Google Scholar]
  26. Persechini A., Stemmer P. M., Ohashi I. Localization of unique functional determinants in the calmodulin lobes to individual EF hands. J Biol Chem. 1996 Dec 13;271(50):32217–32225. doi: 10.1074/jbc.271.50.32217. [DOI] [PubMed] [Google Scholar]
  27. Roberts D. M., Besl L., Oh S. H., Masterson R. V., Schell J., Stacey G. Expression of a calmodulin methylation mutant affects the growth and development of transgenic tobacco plants. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8394–8398. doi: 10.1073/pnas.89.17.8394. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Roberts D. M., Burgess W. H., Watterson D. M. Comparison of the NAD Kinase and Myosin Light Chain Kinase Activator Properties of Vertebrate, Higher Plant, and Algal Calmodulins. Plant Physiol. 1984 Jul;75(3):796–798. doi: 10.1104/pp.75.3.796. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Roberts D. M., Crea R., Malecha M., Alvarado-Urbina G., Chiarello R. H., Watterson D. M. Chemical synthesis and expression of a calmodulin gene designed for site-specific mutagenesis. Biochemistry. 1985 Sep 10;24(19):5090–5098. doi: 10.1021/bi00340a020. [DOI] [PubMed] [Google Scholar]
  30. Roberts D. M., Rowe P. M., Siegel F. L., Lukas T. J., Watterson D. M. Trimethyllysine and protein function. Effect of methylation and mutagenesis of lysine 115 of calmodulin on NAD kinase activation. J Biol Chem. 1986 Feb 5;261(4):1491–1494. [PubMed] [Google Scholar]
  31. Roberts D. M., Zimmer W. E., Watterson D. M. The use of synthetic oligodeoxyribonucleotides in the examination of calmodulin gene and protein structure and function. Methods Enzymol. 1987;139:290–303. doi: 10.1016/0076-6879(87)39093-7. [DOI] [PubMed] [Google Scholar]
  32. Rowe P. M., Wright L. S., Siegel F. L. Calmodulin N-methyltransferase. Partial purification and characterization. J Biol Chem. 1986 May 25;261(15):7060–7069. [PubMed] [Google Scholar]
  33. Sharma R. K., Wang T. H., Wirch E., Wang J. H. Purification and properties of bovine brain calmodulin-dependent cyclic nucleotide phosphodiesterase. J Biol Chem. 1980 Jun 25;255(12):5916–5923. [PubMed] [Google Scholar]
  34. Su Z., Blazing M. A., Fan D., George S. E. The calmodulin-nitric oxide synthase interaction. Critical role of the calmodulin latch domain in enzyme activation. J Biol Chem. 1995 Dec 8;270(49):29117–29122. doi: 10.1074/jbc.270.49.29117. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Takeda T., Imai Y., Yamamoto M. Substitution at position 116 of Schizosaccharomyces pombe calmodulin decreases its stability under nitrogen starvation and results in a sporulation-deficient phenotype. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9737–9741. doi: 10.1073/pnas.86.24.9737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. VanBerkum M. F., Means A. R. Three amino acid substitutions in domain I of calmodulin prevent the activation of chicken smooth muscle myosin light chain kinase. J Biol Chem. 1991 Nov 15;266(32):21488–21495. [PubMed] [Google Scholar]
  38. Watterson D. M., Sharief F., Vanaman T. C. The complete amino acid sequence of the Ca2+-dependent modulator protein (calmodulin) of bovine brain. J Biol Chem. 1980 Feb 10;255(3):962–975. [PubMed] [Google Scholar]
  39. Wright L. S., Bertics P. J., Siegel F. L. Calmodulin N-methyltransferase. Kinetics, mechanism, and inhibitors. J Biol Chem. 1996 May 31;271(22):12737–12743. doi: 10.1074/jbc.271.22.12737. [DOI] [PubMed] [Google Scholar]
  40. Zhang M., Huque E., Vogel H. J. Characterization of trimethyllysine 115 in calmodulin by 14N and 13C NMR spectroscopy. J Biol Chem. 1994 Feb 18;269(7):5099–5105. [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES