Regulation of the p53 tumor suppressor is necessary for preventing excessive gene activity and cell growth. In the absence of cellular stress, the intrinsically disordered protein (IDP) MDMX binds tightly to p53 and inhibits its transactivation and DNA binding activity. This requires CK1α binding MDMX and phosphorylating residue S289, which disrupts an autoinhibitory interaction between the acidic domain (AD) and the p53 binding domain (p53BD) of MDMX. We are investigating the structural and dynamic interactions between MDMX and CK1α by conducting titrations paired with nuclear magnetic resonance (NMR) spectroscopy. Using this approach, we can detect residue specific interactions between MDMX and CK1α at two short linear motifs (SLiMs) in the AD, referred to as WW and WF, and a zinc finger (ZF) domain. The WW and WF motifs play a key role in autoinhibition and bind directly to the p53BD. Upon phosphorylation of S289 by CK1α we observe the appearance of new peaks and large chemical shift changes in MDMX. Our current data suggest that MDMX and CK1α remain tightly bound following phosphorylation and we propose that this complex negatively regulates p53 DNA binding. We are also investigating the role of a conserved linker between the p53BD and AD on autoinhibition. Previous results suggested an orientation of the WW motif consistent with the alpha fold (AF2) model of human MDMX. To test the AF2 model we dramatically shortened the native linker and inserted small GS linkers that were 6, 12, and 18 residues long. Isothermal titration calorimetry (ITC) of the 3xGS-linked MDMX behaves as a super autoinhibitor, suggesting the orientation of the WW motif in the AF2 model is correct. Future studies will focus on MDMX fragments containing the p53BD, AD, and ZF in complex with CK1α and the effects on p53 inhibition.
Gruccio et al. (Sun,) studied this question.