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

S100B is a dimeric Ca2+-binding protein that undergoes a 90 ± 3° 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, Tb3+ 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 Ca2+ ion dissociation. In competition studies with Tb3+, the dissociation rates of Ca2+ (koff) 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 Ca2+ association rate constants for S100B, kon, for the EF2 domains in the absence and presenc...