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.
Markowitz, Joseph, Richard R. Rustandi, Kristen M. Varney, Paul T. Wilder, Ryan Udan, Su Ling Wu, William DeW Horrocks, and David J. Weber. "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." Biochemistry 44, no. 19 (2005): 7305-7314.