Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1999 Nov;8(11):2444–2454. doi: 10.1110/ps.8.11.2444

Conformational and metal-binding properties of androcam, a testis-specific, calmodulin-related protein from Drosophila.

S R Martin 1, A Q Lu 1, J Xiao 1, J Kleinjung 1, K Beckingham 1, P M Bayley 1
PMCID: PMC2144196  PMID: 10595548

Abstract

Androcam is a testis-specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF-hand calcium-binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calcium-free androcam shows a biphasic process characteristic of a two-domain protein, with the apo-N-domain less stable than the apo-C-domain. The two EF hands of the C-domain of androcam bind calcium cooperatively with 40-fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding [Ka(Mg) approximately 3 x 10(3) M(-1)]. Weak calcium binding is also detected at one or more N-domain sites. Compared to apo-calmodulin, apo-androcam has a smaller conformational response to calcium and a lower alpha-helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N-domain of apo-androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium-dependent conformational change in this domain. The affinity of model target peptides for androcam is 10(3)-10(5) times lower than for calmodulin, and interaction of the N-domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium-induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium-binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium-binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N-domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis-specific calcium signaling functions.

Full Text

The Full Text of this article is available as a PDF (438.1 KB).

Selected References

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

  1. Andres A. J., Thummel C. S. The Drosophila 63F early puff contains E63-1, an ecdysone-inducible gene that encodes a novel Ca(2+)-binding protein. Development. 1995 Aug;121(8):2667–2679. doi: 10.1242/dev.121.8.2667. [DOI] [PubMed] [Google Scholar]
  2. Barth A., Martin S. R., Bayley P. M. Specificity and symmetry in the interaction of calmodulin domains with the skeletal muscle myosin light chain kinase target sequence. J Biol Chem. 1998 Jan 23;273(4):2174–2183. doi: 10.1074/jbc.273.4.2174. [DOI] [PubMed] [Google Scholar]
  3. Bayley P. M., Findlay W. A., Martin S. R. Target recognition by calmodulin: dissecting the kinetics and affinity of interaction using short peptide sequences. Protein Sci. 1996 Jul;5(7):1215–1228. doi: 10.1002/pro.5560050701. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Biekofsky R. R., Martin S. R., Browne J. P., Bayley P. M., Feeney J. Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual-site Ca2+ coordination. Biochemistry. 1998 May 19;37(20):7617–7629. doi: 10.1021/bi9800449. [DOI] [PubMed] [Google Scholar]
  5. Bootman M. D., Berridge M. J. The elemental principles of calcium signaling. Cell. 1995 Dec 1;83(5):675–678. doi: 10.1016/0092-8674(95)90179-5. [DOI] [PubMed] [Google Scholar]
  6. Brokaw C. J. Calcium sensors in sea urchin sperm flagella. Cell Motil Cytoskeleton. 1991;18(2):123–130. doi: 10.1002/cm.970180207. [DOI] [PubMed] [Google Scholar]
  7. Brown S. E., Martin S. R., Bayley P. M. Kinetic control of the dissociation pathway of calmodulin-peptide complexes. J Biol Chem. 1997 Feb 7;272(6):3389–3397. doi: 10.1074/jbc.272.6.3389. [DOI] [PubMed] [Google Scholar]
  8. Browne J. P., Strom M., Martin S. R., Bayley P. M. The role of beta-sheet interactions in domain stability, folding, and target recognition reactions of calmodulin. Biochemistry. 1997 Aug 5;36(31):9550–9561. doi: 10.1021/bi970460d. [DOI] [PubMed] [Google Scholar]
  9. Crivici A., Ikura M. Molecular and structural basis of target recognition by calmodulin. Annu Rev Biophys Biomol Struct. 1995;24:85–116. doi: 10.1146/annurev.bb.24.060195.000505. [DOI] [PubMed] [Google Scholar]
  10. Doyle K. E., Kovalick G. E., Lee E., Beckingham K. Drosophila melanogaster contains a single calmodulin gene. Further structure and expression studies. J Mol Biol. 1990 Jun 20;213(4):599–605. doi: 10.1016/S0022-2836(05)80245-1. [DOI] [PubMed] [Google Scholar]
  11. Edelhoch H. Spectroscopic determination of tryptophan and tyrosine in proteins. Biochemistry. 1967 Jul;6(7):1948–1954. doi: 10.1021/bi00859a010. [DOI] [PubMed] [Google Scholar]
  12. Falke J. J., Drake S. K., Hazard A. L., Peersen O. B. Molecular tuning of ion binding to calcium signaling proteins. Q Rev Biophys. 1994 Aug;27(3):219–290. doi: 10.1017/s0033583500003012. [DOI] [PubMed] [Google Scholar]
  13. Findlay W. A., Gradwell M. J., Bayley P. M. Role of the N-terminal region of the skeletal muscle myosin light chain kinase target sequence in its interaction with calmodulin. Protein Sci. 1995 Nov;4(11):2375–2382. doi: 10.1002/pro.5560041116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Findlay W. A., Martin S. R., Beckingham K., Bayley P. M. Recovery of native structure by calcium binding site mutants of calmodulin upon binding of sk-MLCK target peptides. Biochemistry. 1995 Feb 21;34(7):2087–2094. doi: 10.1021/bi00007a001. [DOI] [PubMed] [Google Scholar]
  15. Fyrberg C., Parker H., Hutchison B., Fyrberg E. Drosophila melanogaster genes encoding three troponin-C isoforms and a calmodulin-related protein. Biochem Genet. 1994 Apr;32(3-4):119–135. doi: 10.1007/BF00554420. [DOI] [PubMed] [Google Scholar]
  16. Gill S. C., von Hippel P. H. Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem. 1989 Nov 1;182(2):319–326. doi: 10.1016/0003-2697(89)90602-7. [DOI] [PubMed] [Google Scholar]
  17. Guex N., Peitsch M. C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis. 1997 Dec;18(15):2714–2723. doi: 10.1002/elps.1150181505. [DOI] [PubMed] [Google Scholar]
  18. Hennessey J. P., Jr, Manavalan P., Johnson W. C., Jr, Malencik D. A., Anderson S. R., Schimerlik M. I., Shalitin Y. Conformational transitions of calmodulin as studied by vacuum-uv CD. Biopolymers. 1987 Apr;26(4):561–571. doi: 10.1002/bip.360260409. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Kawasaki H., Kurosu Y., Kasai H., Isobe T., Okuyama T. Limited digestion of calmodulin with trypsin in the presence or absence of various metal ions. J Biochem. 1986 May;99(5):1409–1416. doi: 10.1093/oxfordjournals.jbchem.a135610. [DOI] [PubMed] [Google Scholar]
  21. Kretsinger R. H. Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem. 1980;8(2):119–174. doi: 10.3109/10409238009105467. [DOI] [PubMed] [Google Scholar]
  22. Krueger J. K., Zhi G., Stull J. T., Trewhella J. Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase. Biochemistry. 1998 Oct 6;37(40):13997–14004. doi: 10.1021/bi981311d. [DOI] [PubMed] [Google Scholar]
  23. Linse S., Brodin P., Johansson C., Thulin E., Grundström T., Forsén S. The role of protein surface charges in ion binding. Nature. 1988 Oct 13;335(6191):651–652. doi: 10.1038/335651a0. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Mackall J., Klee C. B. Calcium-induced sensitization of the central helix of calmodulin to proteolysis. Biochemistry. 1991 Jul 23;30(29):7242–7247. doi: 10.1021/bi00243a028. [DOI] [PubMed] [Google Scholar]
  26. Martin S. R., Bayley P. M., Brown S. E., Porumb T., Zhang M., Ikura M. Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule. Biochemistry. 1996 Mar 19;35(11):3508–3517. doi: 10.1021/bi952522a. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Martin S. R., Maune J. F., Beckingham K., Bayley P. M. Stopped-flow studies of calcium dissociation from calcium-binding-site mutants of Drosophila melanogaster calmodulin. Eur J Biochem. 1992 May 1;205(3):1107–1114. doi: 10.1111/j.1432-1033.1992.tb16879.x. [DOI] [PubMed] [Google Scholar]
  29. Maune J. F., Beckingham K., Martin S. R., Bayley P. M. Circular dichroism studies on calcium binding to two series of Ca2+ binding site mutants of Drosophila melanogaster calmodulin. Biochemistry. 1992 Sep 1;31(34):7779–7786. doi: 10.1021/bi00149a006. [DOI] [PubMed] [Google Scholar]
  30. Maune J. F., Klee C. B., Beckingham K. Ca2+ binding and conformational change in two series of point mutations to the individual Ca(2+)-binding sites of calmodulin. J Biol Chem. 1992 Mar 15;267(8):5286–5295. [PubMed] [Google Scholar]
  31. 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]
  32. 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]
  33. Ohki S., Ikura M., Zhang M. Identification of Mg2+-binding sites and the role of Mg2+ on target recognition by calmodulin. Biochemistry. 1997 Apr 8;36(14):4309–4316. doi: 10.1021/bi962759m. [DOI] [PubMed] [Google Scholar]
  34. Olwin B. B., Storm D. R. Calcium binding to complexes of calmodulin and calmodulin binding proteins. Biochemistry. 1985 Dec 31;24(27):8081–8086. doi: 10.1021/bi00348a037. [DOI] [PubMed] [Google Scholar]
  35. Peersen O. B., Madsen T. S., Falke J. J. Intermolecular tuning of calmodulin by target peptides and proteins: differential effects on Ca2+ binding and implications for kinase activation. Protein Sci. 1997 Apr;6(4):794–807. doi: 10.1002/pro.5560060406. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Persechini A., Gansz K. J., Paresi R. J. A role in enzyme activation for the N-terminal leader sequence in calmodulin. J Biol Chem. 1996 Aug 9;271(32):19279–19282. doi: 10.1074/jbc.271.32.19279. [DOI] [PubMed] [Google Scholar]
  37. Smith V. L., Doyle K. E., Maune J. F., Munjaal R. P., Beckingham K. Structure and sequence of the Drosophila melanogaster calmodulin gene. J Mol Biol. 1987 Aug 5;196(3):471–485. doi: 10.1016/0022-2836(87)90025-8. [DOI] [PubMed] [Google Scholar]
  38. Tsai M. D., Drakenberg T., Thulin E., Forsén S. Is the binding of magnesium (II) to calmodulin significant? An investigation by magnesium-25 nuclear magnetic resonance. Biochemistry. 1987 Jun 16;26(12):3635–3643. doi: 10.1021/bi00386a057. [DOI] [PubMed] [Google Scholar]
  39. Tsalkova T. N., Privalov P. L. Thermodynamic study of domain organization in troponin C and calmodulin. J Mol Biol. 1985 Feb 20;181(4):533–544. doi: 10.1016/0022-2836(85)90425-5. [DOI] [PubMed] [Google Scholar]
  40. Török K., Lane A. N., Martin S. R., Janot J. M., Bayley P. M. Effects of calcium binding on the internal dynamic properties of bovine brain calmodulin, studied by NMR and optical spectroscopy. Biochemistry. 1992 Apr 7;31(13):3452–3462. doi: 10.1021/bi00128a020. [DOI] [PubMed] [Google Scholar]
  41. Vriend G. WHAT IF: a molecular modeling and drug design program. J Mol Graph. 1990 Mar;8(1):52-6, 29. doi: 10.1016/0263-7855(90)80070-v. [DOI] [PubMed] [Google Scholar]
  42. 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]
  43. Yaswen P., Smoll A., Hosoda J., Parry G., Stampfer M. R. Protein product of a human intronless calmodulin-like gene shows tissue-specific expression and reduced abundance in transformed cells. Cell Growth Differ. 1992 Jun;3(6):335–345. [PubMed] [Google Scholar]
  44. Yazawa M., Vorherr T., James P., Carafoli E., Yagi K. Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump. Biochemistry. 1992 Mar 31;31(12):3171–3176. doi: 10.1021/bi00127a018. [DOI] [PubMed] [Google Scholar]
  45. de Groot B. L., van Aalten D. M., Scheek R. M., Amadei A., Vriend G., Berendsen H. J. Prediction of protein conformational freedom from distance constraints. Proteins. 1997 Oct;29(2):240–251. doi: 10.1002/(sici)1097-0134(199710)29:2<240::aid-prot11>3.0.co;2-o. [DOI] [PubMed] [Google Scholar]

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

RESOURCES