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 Ca 2+ (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 presence of the p53 peptide are 1.1 × 106 and 3.5 × 105 M-1 s-1, respectively. These two association rate constants are significantly below the diffusion control (∼109 M-1 s-1) and likely involve both Ca2+ ion association and a Ca2+-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.

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