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

• Published in: Biochemistry (Easton) 2005-05, Vol.44 (19), p.7305-7314
• Main Authors: Markowitz, Joseph, Rustandi, Richard R, Varney, Kristen M, Wilder, Paul T, Udan, Ryan, Wu, Su Ling, Horrocks, William DeW, Weber, David J
• Format: Article
• Language: English
• Subjects: Alanine - genetics; Amino Acid Sequence; Animals; Calcium - chemistry; EF Hand Motifs - genetics; Glutamic Acid - genetics; Humans; Macromolecular Substances - chemistry; Macromolecular Substances - metabolism; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Nerve Growth Factors - chemistry; Nerve Growth Factors - genetics; Nerve Growth Factors - metabolism; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - metabolism; Protein Binding - genetics; Protein Structure, Tertiary - genetics; Rats; S100 Calcium Binding Protein beta Subunit; S100 Proteins - chemistry; S100 Proteins - genetics; S100 Proteins - metabolism; Terbium - chemistry; Tumor Suppressor Protein p53 - chemistry; Tumor Suppressor Protein p53 - genetics; Tumor Suppressor Proteins - chemistry; Tumor Suppressor Proteins - genetics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi050321t

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+ (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 Ca2+ association rate constants for S100B, k on, 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.