Role of Asp37 in metal-binding and conformational change of ciliate Euplotes octocarinatus centrin

Wen Liu, Lian Duan, Bing Zhao, Ya-Qin Zhao, Ai-Hua Liang, Bin-Sheng Yang

Institute of Biotechnology, Shanxi Uni-versity, Taiyuan, China.
Institute of Molecular Science, Shanxi University, Taiyuan, China.
Institute of Molecular Science, Shanxi University, Taiyuan, China;.

DOI: 10.4236/health.2010.23037                

Abstract

Centrin is a member of the EF-hand super family that plays critical role in the centrosome duplication and separation. To investigate the role of Asp37 in the process of metal-binding and conformational change of ciliate Euplotes octocarinatus centrin (EoCen), the mutant D37K, in which aspartic acid 37 had been replaced by lysine, was first obtained by the site-directed mutagenesis. Then 2-p-toluidinylnaphthalene-6- sulfonate (TNS) was used as a fluorescence probe to detect the conformational change of the protein. The results show that the metal- binding capability of the site I of EoCen was lost by the mutation of Asp37→Lys. In comparison the Tb3+-saturated EoCen, the hydrophobic surface of D37K, which is exposed by the binding of Tb3+, has shrunk sharply, suggesting that Asp37 plays an important role in maintaining the proper conformation of EoCen in the presence of Tb3+. Meanwhile, the conditional binding constants of TNS with Tb3+-loaded EoCen and D37K were obtained, K(Tb3+-EoCen-TNS)=(7.38 ±0.25)×105 M-1 and K(Tb3+-D37K-TNS)=(1.16± 0.05)×106 M-1.

Keywords

Centrin; Aspartic Acid; Tb3+; TNS

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Thompson, J.R., Ryan, Z.C., Salisbury, J.L. and Kumar, R. (2006) The structure of the human centrin 2-xeroderma pigmentosum group C protein complex. Journal of Biological Chemistry, 281, 18746-18752.
[2]	Lingle, W.L., Lutz, W.H., Ingle, J.N., et al. (1998) Centrosome hypertrophy in human breast tumors: Implications for genomic stability and cell polarity. Proceedings of the National Academy of Sciences USA, 95, 2950-2955.
[3]	Jaspersen, S.L. and Winey, M. (2004) The budding yeast spindle pole body: Structure, duplication, and function. Annual Review of Cell and Developmental Biology, 20, 1-28.
[4]	Lingle, W.L., Barrett, S.L., Negron, V.C., et al. (2002) Centrosome amplification drives chromosomal instability in breast tumor development. Proceedings of the National Academy of Sciences USA, 99, 1978-1983.
[5]	Lingle, W.L. and Salisbury, J.L. (1999) Alter centrosome structure is associated with abnormal mitoses in human breast tumors. American Journal of Pathology, 155, 1941-1951.
[6]	Lingle, W.L. and Salisbury, J.L. (2000) The role of the centrosome in the development of malignant tumors. Current Topics in Developmental Biology, 49, 313-329.
[7]	Yang, A., Miron, S., Mouawad, L., et al. (2006) Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair. Biochemistry, 45, 3653-3663.
[8]	Li, G.T., Wang, Z.J., Zhao, Y.Q., et al. (2007) The spectral studies on the effect of Glu 101 to the metal binding characteristic of Euplotes octocarinatus centrin. Spectrochim Acta A, 67, 1189-1193.
[9]	Wiech, H.B., Geier, M., Paschke, T., et al. (1996) Characterization of green alga, yeast, and human centrins. Journal of Biological Chemistry, 271, 22453-22461.
[10]	Cox, J.A., Tirone, F., Durussel, I., et al. (2005) Calcium and magnesium binding to human centrin 3 and interaction with target peptides. Biochemistry, 44, 840-850.
[11]	Wang, Z.J., Ren, L.X., Zhao, Y.Q., et al. (2007) Metal ions-induced conformational change of P23 by using TNS as fluorescence probe. Spectrochim Acta Part A, 66, 1323-1326.
[12]	Wang, Z.J., Ren, L.X., Zhao, Y.Q., et al. (2008) Investigation on the binding of TNS to centrin: An EF-hand protein. Spectrochim Acta Part A, 70, 892-897.
[13]	Weber, C.V., Lee, D., Chazin, W.J. and Huang, B. (1994) High level expression in Escherichia coli and characterization of the EF-hand calcium-binding protein caltractin. Journal of Biological Chemistry, 269, 15795-15802.
[14]	Rigden, D.J. and Galperin, M.Y. (2004) The DxDxDG Motif for calcium binding: Multiple structural contexts and implications for evolution. Journal of Biological Chemistry, 343, 971-984.
[15]	Hu, J., Jia, X., Li, Q., et al. (2004) Binding of La3+ to calmodulin and its effects on the interaction between calmodulin and calmodulin binding peptide, polistes mastoparan. Biochemistry, 43, 2688-2698.
[16]	Shannon, R.D. (1976) Revised effective ionic radii and systematic studies of interatomic distances in halides and chalcogenides. Acta Crystallographica A, 32, 751-767.
[17]	Bai, H.J., Liu, W. and Yang, B.S. (2002) Fluorescence studies on the kinetics of terbium (III) removal from monoterbium transferrins by tartaric acid. Acta Chimica Sinica, 60, 1253-1257.
[18]	Duan, L., Zhao, Y.Q., Wang, Z.J., et al. (2008) Lutetium (III)-dependent self-assembly study of ciliate Euplotes octocarinatus centrin. Journal of Inorganic Biochemistry, 102, 268-277.
[19]	Pace, C.N., Vajdos, F., Fee, L., et al. (1995) How to measure and predict the molar absorption coefficient of a protein. Protein Science, 4, 2411-2423.
[20]	McClure, W.O. and Edelman, G.M. (1966) Fluorescent probes for conformational states of proteins. I. Mechanism of fluorescence of 2-p-Toluidinylnaphthalene-6-sulfonate: A hydrophobic probe. Biochemistry, 5, 1908-1919.
[21]	Durussela, I., Blouquitb, Y., Middendorpc, S., et al. (2000) Cation- and peptide-binding properties of human centrin 2. FEBS Letters, 472, 208-212.
[22]	Ikura, M., Clore, G.M., Gronenborn, A.M., et al. (1992) Solution structure of a calmodulin-target peptide complex by multidimensional NMR. Bax Science, 256, 632-638.
[23]	Zhang, M., Tanaka, T. and Ikura, M. (1995) Calcium- induced conformational transition revealed by the solution structure of apo calmodulin. Nature Structural Biology, 2, 758-767.
[24]	Wang, Z.J., Zhao, Y.Q., Ren, L.X., et al. (2007) Spectral study on the interaction of ciliate Euplotes octocarinatus centrin and metal ions. Journal of Photochemistry and Photobiology A, 186, 178-186.
[25]	Hu, H.T. and Chazin, W.J. (2003) Unique features in the C-terminal domain provide caltractin with target specificity. Journal of Biological Chemistry, 330, 473-484.
[26]	Tourbez, M., Firanescu, C., Yang, A., et al. (2004) Calcium-dependent self-assembly of human centrin 2. Journal of Biological Chemistry, 279, 47672-47680.
[27]	Ren, L. X., Zhao, Y.Q., Feng, J.Y., et al. (2006) Terbium- and calcium-binding properties of N-terminal domain of Euplotes centrin. Chinese Journal of Inorganic Chemistry, 22, 87-90.
[28]	Yang, B.S., Liu, W., Ren, L.X., et al. (2009) The spectral studies on the metal binding characteristic of N-terminal domain of ciliate Euplotes octocarinatus centrin. Journal of Shanxi University (Nature and Science Edition), 32(4), 572-576.
[29]	Yang, B.S., Yang, P. and Song, L.H. (1984) The binding of Gd (III) to human serum albumin. Chinese Science Bulletin, 29, 1502-1505.