Characterization of the interaction of calcyclin (S100A6) and calcyclin-binding protein.

Nowotny M, Bhattacharya S, Filipek A, Krezel AM, Chazin W, Kuznicki J
J Biol Chem. 2000 275 (40): 31178-82

PMID: 10884380 · DOI:10.1074/jbc.M001622200

Calcyclin (S100A6) is an S100 calcium-binding protein whose expression is up-regulated in proliferating and differentiating cells. A novel 30-kDa protein exhibiting calcium-dependent calcyclin-binding (calcyclin-binding protein, CacyBP) had been identified, purified, and cloned previously (Filipek, A., and Kuznicki, J. (1998) J. Neurochem. 70, 1793-1798). Here, we have defined the calcyclin binding region using limited proteolysis and a set of deletion mutants of CacyBP. A fragment encompassing residues 178-229 (CacyBP-(178-229)) was capable of full binding to calcyclin. CacyBP-(178-229) was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified. The protein fragment cleaved from the glutathione S-transferase fusion protein was shown by CD to contain 5% alpha-helix, 15% beta -sheet, and 81% random coil. Fluorescence spectroscopy was used to determine calcyclin dissociation constants of 0.96 and 1.2 microm for intact CacyBP and CacyBP-(178-229), respectively, indicating that the fragment can be used for characterization of calcyclin-CacyBP interactions. NMR analysis of CacyBP-(178-229) binding-induced changes in the chemical shifts of (15)N-enriched calcyclin revealed that CacyBP binding occurs at a discrete site on calcyclin with micromolar affinity.

MeSH Terms (21)

Amino Acid Sequence Binding Sites Calcium-Binding Proteins Cell Cycle Proteins Chromatography, Affinity Circular Dichroism Cloning, Molecular DNA Primers Escherichia coli Gene Deletion Glutathione Transferase Magnetic Resonance Spectroscopy Models, Genetic Molecular Sequence Data Mutagenesis, Site-Directed Protein Binding Recombinant Fusion Proteins S100 Calcium Binding Protein A6 S100 Proteins Spectrometry, Fluorescence Up-Regulation

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