Activation of p53: How phosphorylated Ser15 triggers sequential phosphorylation of p53 at Thr18 by CK1$

The N-terminal transactivation domain (TAD) of p53 is a disordered region with multiple phosphorylation sites. Phosphorylation at Thr18 is crucial for the release of p53 from its negative regulator, MDM2. In stressed cells, CK1$ is responsible for phosphorylating Thr18, but requires Ser15 to be phosphorylated. To understand the mechanistic underpinnings of this sequential phosphorylation, molecular modeling and molecular dynamics simulation studies of these phosphorylation events were carried out. Our models suggest that a positively charged region on CK1$ near the adenosine triphosphate (ATP) binding pocket, which is conserved across species, sequesters the negatively charged pSer15, thereby constraining the positioning of the rest of the peptide, such that the side chain of Thr18 is positioned close to the $-phosphate of ATP. Furthermore, our studies show that the phosphorylated p53 TAD1 (p53pSer15) peptide binds more strongly to CK1$ than does p53. p53 adopts a helical structure when bound to CK1$, which is lost upon phosphorylation at Ser15, thus gaining higher flexibility and ability to morph into the binding site. We propose that upon phosphorylation at Ser15 the p53 TAD1 peptide binds to CK1$ through an electrostatically driven induced fit mechanism resulting in a flanking fuzzy complex.