We investigated the ways S100B, S100A1, S100A2, S100A4, and S100A6 bind to the different oligomeric forms of the tumor suppressor p53 in vitro, using analytical ultracentrifugation and multiangle light scattering. It is established that members of the S100 protein family bind to the tetramerization domain (residues 325-355) of p53 when it is uncovered in the monomer, and so binding can disrupt the tetramer. We found a stoichiometry of one dimer of S100 bound to a monomer of p53. We discovered that some S100 proteins could also bind to the tetramer. S100B bound the tetramer and also disrupted the dimer by binding monomeric p53. S100A2 bound monomeric p53 as well as tetrameric, whereas S100A1 only bound monomeric p53. S100A6 bound more tightly to tetrameric than to monomeric p53. We also identified an additional binding site for S100 proteins in the transactivation domain (1-57) of p53. Based on our results and published observations in vivo, we propose a model for the binding of S100 proteins to p53 that can explain both activation and inhibition of p53-mediated transcription. Depending on the concentration of p53 and the member of the S100 family, binding can alter the balance between monomer and tetramer in either direction.The S100 family is a highly conserved group of more than 20 members of small, acidic calcium-binding proteins in vertebrates (1). They are called S100 because they remain soluble in 100% ammonium sulfate at neutral pH (2). S100 proteins are dimers or form higher oligomers (3, 4). They have intracellular functions such as the regulation of protein phosphorylation, the regulation of calcium homeostasis, cell survival, proliferation, and differentiation, as well as extracellular functions, for example, as attractors for leukocytes and macrophages, neurite outgrowth, or the induction of apoptosis (5-8). Further, the expression of several S100 proteins has been linked to metastasis (9) and different kinds of melanomas and carcinomas (8). Nevertheless, the molecular mechanism of action of the S100 proteins is not fully understood.The tumor suppressor p53 is a crucial factor in the development of cancer. It acts as the central inducer of apoptosis and cell cycle arrest (10, 11). Posttranslational modifications and interaction with proteins regulate its activity (12)(13)(14). The interaction with the tumor suppressor protein p53 is a common feature of the S100 proteins (15-19). We previously demonstrated that S100 proteins generally bind to the tetramerization domain (residues 325-355) of p53, whereas only a subset can bind its negative regulatory domain (residues 367-393) (16,20). S100B, S100A2, S100A4, and S100A6 have been reported to influence p53-mediated transcription, but the effect remains controversial because some studies show a stimulating effect, whereas others claim that S100 proteins inhibit the transcriptional activity of p53 (17-19, 21, 22). We previously showed that oligomerization of p53 weakens the binding to S100B and S100A4, and it was deduced that S100 proteins inhibit the oligo...

No abstract

S100 proteins modulate p53 activity by interacting with its tetramerization (p53TET, residues 325-355) and transactivation (residues 1-57) domains. In this study, we characterized biophysically the binding of S100A1, S100A2, S100A4, S100A6 and S100B to homologous domains of p63 and p73 in vitro by fluorescence anisotropy, analytical ultracentrifugation and analytical gel filtration. We found that S100A1, S100A2, S100A4, S100A6 and S100B proteins bound different p63 and p73 tetramerization domain variants and naturally occurring isoforms with varying affinities in a calcium-dependent manner. Additional interactions were observed with peptides derived from the p63 and p73 N-terminal transactivation domains. Importantly, S100 proteins bound p63 and p73 with different affinities in their different oligomeric states, similarly to the differential modes of binding to p53. On the basis of our data, we hypothesize that S100 proteins regulate the oligomerization state of all three p53 family members and their isoforms, with a potential physiological relevance in developmental and disease-related processes. The regulation of the p53 family by S100 is complicated and depends on the target preference of each individual S100 protein, the concentration of the proteins and calcium, as well as the splicing variation of p63 or p73. Our results outlining the complexity of the interaction should be considered when studying the functional effects of S100 proteins in their biological context.

Keywords: S100 p53 MDM2 Ternary complex Protein-protein interaction a b s t r a c t S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro.