Structural changes in the C-terminus of Ca²⁺-bound rat S100B(ββ) upon binding to a peptide derived from the C-terminal regulatory domain of p53

Abstract

S100B(ββ) is a dimeric Ca²⁺-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(ββ) in a Ca²⁺-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca²⁺-loaded S100B(ββ) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, ³J_NH-Hα coupling constants, and chemical shift index data show that, like apo- and Ca²⁺-bound S100B(ββ), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2–Arg20; helix 2, Lys29–Asn38; helix 3, Gln50–Asp61; helix 4, Phe70–Phe87), four loops (loop 1, Glu21–His25; loop 2, Glu39–Glu49; loop 3, Glu62–Gly66; loop 4, Phe88–Glu91), and two β-strands (β-strand 1, Lys26–Lys28; β-strand 2, Glu67–Asp69), which forms a short antiparallel β-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca²⁺-bound S100B(ββ). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca²⁺-bound S100B(ββ). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(ββ) support the notion that the p53 peptide binds in a region of S100B(ββ), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.