title: 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.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, Maryland 21201, USA.
authors.forename: Joseph
authors.initials: J
authors.lastname: Markowitz
authors.forename: Richard R
authors.initials: RR
authors.lastname: Rustandi
authors.forename: Kristen M
authors.initials: KM
authors.lastname: Varney
authors.forename: Paul T
authors.initials: PT
authors.lastname: Wilder
authors.forename: Ryan
authors.initials: R
authors.lastname: Udan
authors.forename: Su Ling
authors.initials: SL
authors.lastname: Wu
authors.forename: William DeW
authors.initials: WD
authors.lastname: Horrocks
authors.forename: David J
authors.initials: DJ
authors.lastname: Weber
mesh_terms: D000409:Alanine / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000737:chemistry*; D020832:EF Hand Motifs / Q000235:genetics*; D018698:Glutamic Acid / Q000235:genetics; D006801:Humans; D046911:Macromolecular Substances / Q000737:chemistry / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051381:Rats; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013725:Terbium / Q000737:chemistry; D016159:Tumor Suppressor Protein p53 / Q000737:chemistry* / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000235:genetics
chemical_list: D046911:Macromolecular Substances; D009414:Nerve Growth Factors; D010446:Peptide Fragments; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; C579160:S100B protein, human; C579158:S100b protein, rat; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D013725:Terbium; D018698:Glutamic Acid; D000409:Alanine; D002118:Calcium