Calcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53.

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TitleCalcium-binding properties of wild-type and EF-hand mutants of S100B in the presence and absence of a peptide derived from the C-terminal negative regulatory domain of p53.
Publication TypeJournal Article
Year of Publication2005
AuthorsMarkowitz, J, Rustandi, RR, Varney, KM, Wilder, PT, Udan, R, Wu, SLing, Horrocks, WDeW, Weber, DJ
JournalBiochemistry
Volume44
Issue19
Pagination7305-14
Date Published2005 May 17
ISSN0006-2960
KeywordsAlanine, Amino Acid Sequence, Animals, Calcium, EF Hand Motifs, Glutamic Acid, Humans, Macromolecular Substances, Magnetic Resonance Spectroscopy, Molecular Sequence Data, Nerve Growth Factors, Peptide Fragments, Protein Binding, Protein Structure, Tertiary, Rats, S100 Proteins, Terbium, Tumor Suppressor Protein p53, Tumor Suppressor Proteins
Abstract

S100B is a dimeric Ca(2+)-binding protein that undergoes a 90 +/- 3 degrees rotation of helix 3 in the typical EF-hand domain (EF2) upon the addition of calcium. The large reorientation of this helix is a prerequisite for the interaction between each subunit of S100B and target proteins such as the tumor suppressor protein, p53. In this study, Tb(3+) was used as a probe to examine how binding of a 22-residue peptide derived from the C-terminal regulatory domain of p53 affects the rate of Ca(2+) ion dissociation. In competition studies with Tb(3+), the dissociation rates of Ca(2+) (k(off)) from the EF2 domains of S100B in the absence and presence of the p53 peptide was determined to be 60 and 7 s(-)(1), respectively. These data are consistent with a previously reported result, which showed that that target peptide binding to S100B enhances its calcium-binding affinity [Rustandi et al. (1998) Biochemistry 37, 1951-1960]. The corresponding Ca(2+) association rate constants for S100B, k(on), for the EF2 domains in the absence and presence of the p53 peptide are 1.1 x 10(6) and 3.5 x 10(5) M(-)(1) s(-)(1), respectively. These two association rate constants are significantly below the diffusion control ( approximately 10(9) M(-)(1) s(-)(1)) and likely involve both Ca(2+) ion association and a Ca(2+)-dependent structural rearrangement, which is slightly different when the target peptide is present. EF-hand calcium-binding mutants of S100B were engineered at the -Z position (EF-hand 1, E31A; EF-hand 2, E72A; both EF-hands, E31A + E72A) and examined to further understand how specific residues contribute to calcium binding in S100B in the absence and presence of the p53 peptide.

DOI10.1021/bi050321t
Alternate JournalBiochemistry
PubMed ID15882069
Grant ListF30-NS043916 / NS / NINDS NIH HHS / United States
GM58888 / GM / NIGMS NIH HHS / United States