S100B regulation of p53 phosphorylation by PKC(alpha) and structural characterization of zinc(2+)-binding to calcium(2+)-S100B

Abstract

The S100B protein is a small acidic metal binding protein that has been found to be associated with neurological disorders and traumas, diabetes, and cancer. This work examines Zn2+ regulation of S100B Ca 2+-binding affinity and target protein interaction and also explores S100B interaction and regulation of an important target protein, tumor suppressor p53. It was found that S100B binds p53 preventing PKC phosphorylation of p53 (1), and this interaction connects the molecular and cellular functions of S100B with clinical data that associates S100B expression to several forms of cancer. In order to further characterize this S100B inhibition of PKC activities, peptides based on the PKC phosphorylation domains of p53 (residues 367--388), neuromodulin (residues 37--53), and the regulatory domain of PKC (residues 19--31) were synthesized and shown to be substrates for PKC and the catalytic domain of PKC, PKM, which lacks the Ca2+ and lipid regulatory region of PKC. It was necessary to use PKM in order to separate the influence of Ca2+ on the activation S100B from its effects on PKC allowing kinetic parameters of the PKC-dependent phosphorylation and its Ca2+-dependent inhibition by S100B to be determined for the peptides. While Ca2+-binding appears to be necessary for target protein binding by S100B, Zn2+-binding potentiates both S100B Ca2+-binding affinity and its interaction with certain target proteins. In order to determine how structural changes in S100B upon Zn2+ binding regulate its function, the high resolution structures of the Zn2+-Ca2+-S100B was completed using multi-dimensional heteronuclear NMR experiments. Studies were done to determine the amino acid ligands used for coordinating Zn2+ in S100B revealing what appears to be a conserved Zn2+/Cu2+-binding site at the dimer interface found in several S100 proteins. The structure shows that Zn2+-binding induces rearrangement of the Ca 2+-binding EF-hands in S100B that may contribute to the change in Ca2+-binding affinity found in S100B. Furthermore, there is an extension of the final helix IV and orienting of amino acids involved in target protein interaction upon Zn2+-binding by Ca2+ S100B that may influence target protein affinity.