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Identification of cyclized calmodulin antagonists from a phage display random peptide library

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Summary

To isolate peptide ligands that bound calmodulin (CaM) specifically, we screened an M13 phage library displaying cyclized octamer random peptides with immobilized bovine CaM. Isolates were recovered, sequenced, and deduced to express nine independent peptides, five of which contained the sequence Trp-Gly-Lys (WGK). Four of the nine peptide sequences were synthesized in cyclized, biotinylated form. All of the peptides required Ca2+ to bind CaM. The cyclized, disulfide-bonded form of one such peptide, SCLRWGKWSNCGS, bound CaM better than its reduced form or an analogue in which the cysteine residues were replaced by serine. The cyclized peptide also exhibited the ability to inhibit CaM-dependent kinase activity. Systematic alanine substitution of residues in this peptide sequence implicate the tryptophan residue as being critical for binding, with other residues contributing to binding to varying degrees. Cloning of ligand targets (COLT) confirmed the specificity of one of the cyclized peptides, yielding full-length and C-terminal CaM clones, in addition to a full-length clone of troponin C, a CaM-related protein. This study has demonstrated that conformationally constrained peptides isolated from a phage library acted as specific, Ca2+-dependent CaM ligands.

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References

  1. Kay, B.K.,Biologically displayed random peptides as reagents in mapping protein-protein interactions, Perspect. Drug Discov. Design, 2 (1995) 251–268.

    Google Scholar 

  2. Cwirla, S.E., Peters, E.A., Barrett, R.W. and Dower, W.J.,Peptides on phage: A vast library of peptides for identifying ligands, Proc. Natl. Acad. Sci. USA, 87 (1990) 6378–6382.

    Google Scholar 

  3. Kay, B.K., Adey, N.B., He, Y.S., Manfredi, J.P., Mataragnon, A.H. and Fowlkes, D.M.,An M13 phage library displaying random 38-amino-acid peptides as a source of novel sequences with affinity to selected targets, Gene, 128 (1993) 59–65.

    Google Scholar 

  4. Motti, C., Nuzzo, M., Meola, A., Galfre, G., Felici, F., Cortese, R., Nicosia, A. and Monaci, P.,Recognition by human sera and immunogenicity of HBsAg mimotopes selected from an M13 phage display library, Gene, 146 (1994) 191–198.

    Google Scholar 

  5. Scott, J.K. and Smith, G.P.,Searching for peptide ligands with an epitope library, Science, 249 (1990) 386–390.

    Google Scholar 

  6. Koivunen, E., Wang, B. and Ruoslahti, E.,Isolation of a highly specific ligand for the alpha-5 beta-1 integrin from a phage display library, J. Cell Biol., 124 (1994) 373–380.

    Google Scholar 

  7. Dedman, J.R., Kaetzel, M.A., Chan, H.C., Nelson, D.J. and Jamieson, G.J.,Selection of targeted biological modifiers from a bacteriophage library of random peptides. The identification of novel calmodulin-regulatory peptides, J. Biol. Chem., 268 (1993) 23025–23030.

    Google Scholar 

  8. Sparks, A.B., Quilliam, L.A., Thorn, J.M., Der, C.J. and Kay, B.K.,Identification and characterization of Src SH3 ligands from phage-displayed random peptide libraries, J. Biol. Chem., 269 (1994) 23853–23856.

    Google Scholar 

  9. Munier, H., Blanco, F.J., Precheur, B., Diesis, E., Nieto, J.L., Craescu, C.T. and Barzu, O.,Characterization of a synthetic calmodulin-binding peptide derived from Bacillus anthracis adenylate cyclase, J. Biol. Chem., 268 (1993) 1695–1701.

    Google Scholar 

  10. Newsholme, P., Angelos, K.L. and Walsh, D.A.,High- and intermediate-affinity calmodulin-binding domains of the alpha- and betasubunits of phosphorylase kinase and their potential role in phosphorylation-dependent activation of the holoenzyme, J. Biol. Chem., 267 (1992) 810–818.

    Google Scholar 

  11. Payne, M.E., Fong, Y.L., Ono, T., Colbran, R.J., Kemp, B.E., Soderling, T.R. and Means, A.R.,Calciumcallmodulin-dependent protein kinase II. Characterization of distinct calmodulin binding and inhibitory domains, J. Biol. Chem., 263 (15) (1988) 7190–7195.

    Google Scholar 

  12. Means, A.R., Bagchi, I.C., VanBerkum, M.F. and Kemp, B.E.,Regulation of smooth-muscle myosin light-chain kinase by calmodulin. [Review], Adv. Exp. Med. Biol., 304 (1991) 11–24.

    Google Scholar 

  13. O'Neil, K.T. and DeGrado, W.F.,How calmodulin binds its targets: Sequence-independent recognition of amphiphilic alpha-helices [Review], Trends Biochem. Sci., 15 (1990) 59–64.

    Google Scholar 

  14. Chapman, E.R., Alexander, K., Vorherr, T., Carafoli, E. and Storm, D.R.,Fluorescence energy-transfer analysis of calmodulinpeptide complexes, Biochemistry, 31 (1992) 12819–12825.

    Google Scholar 

  15. Ikura, M., Clore, G.M., Gronenborn, A.M., Zhu, G., Klee, C.B. and Bax, A.,Solution structure of a calmodulin-target peptide complex by multidimensional NMR, Science, 256 (1992) 632–638.

    Google Scholar 

  16. Meador, W.E., Means, A.R. and Quiocho, F.A.,Target enzyme recognition by calmodulin: 2.4 Å Structure of a calmodulin-peptide complex, Science, 257 (1992) 1251–1255.

    Google Scholar 

  17. Roth, S.M., Schneider, D.M., Strobel, L.A., Van, B.M., Means, A.R. and Wand, A.J.,Characterization of the secondary structure of calmodulin in complex with a calmodulin-binding domain peptide, Biochemistry, 31 (1992) 1443–1451.

    Google Scholar 

  18. Ohya, Y. and Botstein, D.,Diverse essential functions revealed by complementing yeast calmodulin mutants, Science, 263 (1994) 963–966.

    Google Scholar 

  19. VanBerkum, M.F. and 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., 266 (1991) 21488–21495.

    Google Scholar 

  20. Adey, N.B. and Kay, B.K.,Identification of calmodulin-binding peptide consensus sequences from a phage-displayed random peptide library, Gene, 169 (1996) 133–134.

    Google Scholar 

  21. Koivunen, E., Wang, B. and Ruoslahti, E.,Phage libraries displaying cyclic peptides with different ring sizes: Ligand specificities of the RGD-directed integrins, Biotechnology, 13 (1995) 265–270.

    Google Scholar 

  22. O'Neil, K.T., Hoess, R.H., Jackson, S.A., Ramachandran, N.S., Mousa, S.A. and DeGrado, W.F.,Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage peptide library, Proteins, 14 (1992) 509–515.

    Google Scholar 

  23. Malencik, D.A. and Anderson, S.R.,Binding of simple peptides, hormones, and neurotransmitters by calmodulin, Biochemistry, 21 (1982) 3480–3486.

    Google Scholar 

  24. Adey, N.B., Sparks, A.B. and Beasley, J.R.,Construction of phage random peptide libraries, In Kay, B.K., Winter, J. and McCafferty, J. (Eds.) Phage Display of Peptides and Protein Domains: A Laboratory Manual, Academic Press, San Diego, CA, in press.

  25. Fowlkes, D.M., Adams, M.D., Fowler, V.A. and Kay, B.K.,Multipurpose vectors for peptide expression on the M13 viral surface, Biotechniques, 13 (1992) 422–428.

    Google Scholar 

  26. Sparks, A.B., Adey, N.B., Quilliam, L.A., Thorn, J.M. and Kay, B.K.,Screening phage-displayed random peptide libraries for SH3 ligands, Methods Enzymol., 255 (1995) 498–509.

    Google Scholar 

  27. Sparks, A.B., Hoffman, N.G., McConnell, S.J., Fowlkes, D.M. and Kay, B.K.,Cloning of ligand targets: Systematic isolation of SH3 domain-containing proteins, Nat. Biotechnol., 14 (1996) 741–744.

    Google Scholar 

  28. Apel, E.D., Byford, M.F., Au, D., Walsh, K.A. and Storm, D.R.,Identification of the protein kinase C phosphorylation site in neuromodulin, Biochemistry, 29 (1990) 2330–2335.

    Google Scholar 

  29. Baudier, J., Deloulme, J.C., Van, D.A., Black, D. and Matthes, H.W,Purification and characterization of a brain-specific protein kinase C substrate, neurogranin (p17). Identification of a consensus amino acid sequence between neurogranin and neuromodulin (GAP43) that corresponds to the protein kinase C phosphorylation site and the calmodulin-binding domain, J. Biol. Chem., 266 (1991) 229–237.

    Google Scholar 

  30. Urbauer, J.L., Short, J.H., Dow, L.K. and Wand, A.J.,Structural analysis of a novel interaction by calmodulin: High-affinity binding of a peptide in the absence of calcium, Biochemistry, 34 (1995) 8099–8109.

    Google Scholar 

  31. James, P., Vorherr, T. and Carafoli, E.,Calmodulin-binding domains: Just two faced or multi-faceted?, Trends Biochem. Sci., 20 (1995) 38–42.

    Google Scholar 

  32. Vandonselaar, M., Hickie, R.A., Quail, J.W. and Delbaere, L.T.,Trifluoperazine-induced conformational change in Ca 2+ -calmodulin, Nat. Struct. Biol., 1 (1994) 795–801.

    Google Scholar 

  33. Giebel, L.B., Cass, R.T., Milligan, D.L., Young, D.C., Arze, R. and Johnson, C.R.,Screening of cyclic peptide phage libraries identifies ligands that bind Streptavidin with high affinities, Biochemistry, 34 (1995) 15430–15435.

    Google Scholar 

  34. Koivunen, E., Gay, D.A. and Ruoslahti, E.,Selection of peptides binding to the alpha-5 beta-1 integrin from phage display library, J. Biol. Chem., 268 (1993) 20205–20210.

    Google Scholar 

  35. McLafferty, M.A., Kent, R.B., Ladner, R.C. and Markland, W.,M13 bacteriophage displaying disulfide-constrained microproteins, Gene, 128 (1993) 29–36.

    Google Scholar 

  36. Clore, G.M., Bax, A., Ikura, M. and Gronenborn, A.M.,Structure of calmodulin-target peptide complexes, Curr. Opin. Struct. Biol., 3 (1993) 838–845.

    Google Scholar 

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Authors and Affiliations

  1. Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, 27599, Chapel Hill, NC, USA

    Heather Hanson Pierce & Brian K. Kay

  2. Department of Biology, University of North Carolina at Chapel Hill, 27599, Chapel Hill, NC, USA

    Brian K. Kay

  3. Myriad Genetics, 84108, Salt Lake City, UT, USA

    Nils Adey

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  1. Heather Hanson Pierce
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  2. Nils Adey
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  3. Brian K. Kay
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Pierce, H.H., Adey, N. & Kay, B.K. Identification of cyclized calmodulin antagonists from a phage display random peptide library. Mol Divers 1, 259–265 (1996). https://doi.org/10.1007/BF01715530

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  • DOI: https://doi.org/10.1007/BF01715530

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