Characterization of the interaction of calcyclin (S100A6) and calcyclin-binding protein.
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.
Nowotny
M
M
Department of Molecular and Cellular Neurobiology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland.
Bhattacharya
S
S
Filipek
A
A
Krezel
A M
AM
Chazin
W
W
Kuznicki
J
J
D000595:Amino Acid Sequence; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D018797:Cell Cycle Proteins; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D015854:Up-Regulation
C491778:CACYBP protein, human; D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D000075383:S100 Calcium Binding Protein A6; D009418:S100 Proteins; C489134:S100a6 protein, mouse; D005982:Glutathione Transferase