Generation and characterization of calmodulin-DHFR sandwich fusion protein

  • Han, Chang Hoon (College of Veterinary Medicine, Cheju National University)
  • Accepted : 2008.09.03
  • Published : 2008.12.01


A calmodulin-dihydrofolate reductase (DHFR) sandwich fusion protein was generated by insertion of calmodulin into the $\beta$-bulge region of DHFR to observe the effects of structurally constraining the calmodulin structure. The calcium binding properties of the sandwich protein were almost identical to calmodulin. Similar to calmodulin ($10.7 {\mu}M$), the sandwich protein bound four equivalents of calcium, with half saturation ($K_{0.5}$) observed at a [$Ca^{2+}$] of $8{\mu}M$. However, nicotinamide adenine dinucleotide (NAD) kinase activation property of the sandwich protein was lower than that of calmodulin. The sandwich protein activated NAD kinase, but to only half of the level obtained with calmodulin. The K 0.5 for both calmodulin and the sandwich protein were approximately the same (1-2 nM). Methylation analyses of the sandwich protein show that insertion of calmodulin into DHFR results in a large decrease in methylation. The $V_{max}$ observed with the sandwich protein (95 nmole/min/ml) was only 22% of the value observed with calmodulin (436 nmol/min/ml) in the presence of calcium. Addition of trimethoprim to the reaction significantly inhibited the observed methylation rate. Overall, the data suggest that the insertion of calmodulin into the DHFR structure has little effect on calcium binding by the individual lobes of calmodulin, but may constrain the lobes in a manner that results in altered interaction with the calmodulin-dependent proteins, and severely perturbed the methyltransferase recognition site.


  1. Bolin JT, Filman DJ, Mattews DA, Hamlin RD, Kraut J. Crysul structures of Escherichia coli and Lactobacillus casei dihydrofolale reductase refined at 1.7 A resolution. I. General features and binding of methotrexate. J Biol Chem 1982, 257, 13650-13662
  2. Colowiek SP, Womack FC. Binding of diffusible molecules by macromolecules: rapid measurement by rate of dialysis. J Biol Chem 1969, .244, 774-777
  3. Craig TA. Wanerson DM, Prendergast FG, Haiech J, Robertll DM. Site-specific mutagenesis of the alphahelices of calmodulin. Effects of altering a charge cluster in the helix that links the two halves of calmodulin. J Biol Chem 1987, 262, 3278-3284
  4. Croueb TH, Klee CB, Positive cooperative binding of calcium to bovine brain calmodulin. Biochemistry 1980, 19, 3692-3698
  5. Filman DJ, Bolin JT, Malthews DA, Kraut J. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. II. Envirorunent of bound NADPH and implications for catalysis. J Biol Chem 1982. 257, 13663-13672
  6. Finn BE. Orakenberg T. Forsen S. The structure of apo-calmodulin. A 1H NMR examination of the carboxy-terminal domain. FEBS Lett 1993, 336, 368- 374
  7. Finn BE, Evenas J, Drakenberg T, Waltho JP, Thulin E, Forsen S. Calcium-induced structural changes and domain autonomy in calmodulin. Nat Struct Biol 1995, 2, 777-783
  8. Haiech J, Klee CB, Demaille JG. Effects of cations on affinity of calmodulin for calcium: ordered binding of calcium ions allows the specific activation of calmodulin-stimulated enzymes. Biochemistry 1981, 20, 3890-3897
  9. Haiech J, Vallet B, Aquaron R, Demaille JG. Ligand binding to macromolecules: determination of binding parameters by combined use of ligand buffers and flow dialysis; application to calcium-binding proteins. Anal Biochem 1980, 105, 18-23
  10. Han CH, Richardson J , Oh SH, Roberts DM. Isolation and kinetic characterization of the calmodulin methyltransrerase from sheep brain. Biochemistry 1993, 32, 13974-13980
  11. Ikura M, Clore GM, Gronenborn AM, Zhu G, Klee CB, Bax A. Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Science 1992, 256, 632-638
  12. 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
  13. Linse S. Helmersson A, Forsen S. Calcium binding to calmodulin and its globular domains. J Biol Chem 1991, 266, 8050-8054
  14. Meador WE. Means AR, Quiocho FA. Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science 1992, 257, 1251-1255
  15. Meador WE, Means AR, Qukxho FA. Modulation of calmodulin plastieity in molecular recognition on the basis of x-ray structures. Science 1993, 262, 1718-1721
  16. Minowa O. Yagi K. Calcium binding to tryptic fragments of calmodulin. J Biochem 1984, 96, 1175-1182
  17. Oh SH, Roberts DM. Analysis of the state of posnranslational calmodulin methylation in developing pea plants. Plant Physiol 1990, 93, 880-887
  18. O'Neil KT, DeGrado WF. How calmodulin binds its targets: sequence independent recognition of amphiphitic alpha-helices. Trends Biochem Sci 1990, 15, 59-64
  19. Porumb T. Determination of calcium-binding constants by flow dialysis. Anal Biochem 1994, 220, 227-237
  20. Richardson JS, Getzoff ED, Richardson DC. The beta bulge: a common small unit of oonrepetitivc protein structure. Proc Natl Acad Sei USA 1978, 75, 2574-2578
  21. Roberts DM, Crea R, Malecha M, Alvarado-Urbina G, Chiarello RH, Wattenon DM. Chemical synthesis and expression of a calmodulin gene designed for sitespecific mutagenesis. Biochemistry 1985, 24, 5090-5098
  22. Roberts DM, Zimmer WE, Watterson DM. The use of synthetic oligodooxyribonucleotides in the examination of caJmodulin gene and protein structure and function. Methods Enzymol 1987, 139, 290-303
  23. Rowe PM, Murtaugb TJ, Bazari WL, Clarke M, Siegel FL. Radiometric assay of S-adenosylmethionine: calmodulin(lysine)N-methyltransferase by calcium-dependent hydrophobic interaction chromatography. Anal Biochem 1983, 133, 394-400
  24. Sondek J, Shortle D. Accommodation of single amino acid insertions by the native state of staphylococcal nuclease. Proteins 1990, 7, 299-305
  25. Sondek J, Shortle D. Structural and energetic differences between insertions and substiMions in staphylococcal nuclease. Proteins 1992, 13, 132-140
  26. Tartof KD, Hobbs CA. New cloning vectors and techniques for easy and rapid restriction mapping. Gene 1988, 67, 169-182
  27. Volz KW, Mattbews DA, Aiden RA. Freer ST, Hansch C, Kaufman BT, Knaut J. Ctystal structure of avian dihydrofolate reductase containing phenyltriazine and NADPH. J Biol Chem 1982, 257, 2528-2536