Allosteric Regulation of MDMX by Protein Disorder

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MDMX and MDM2 are homologues that regulate the p53 tumor suppressor using different mechanisms. While MDM2 is responsible for controlling p53 degradation, MDMX regulates p53 DNA binding and transcriptional activities. MDMX overexpression occurs in a subset of human tumors leading to inactivation of p53. Efficient p53 response to stress and DNA damage involves phosphorylation of MDMX followed by release of p53. Successful targeting of the p53 pathway for cancer therapy requires understanding how MDMX regulates p53 function. MDMX contains an ordered p53 binding domain and RING domain, and two long disordered regions that regulate p53 and CK1α binding in a phosphorylation- dependent manner. Our work identified intra- and intermolecular interactions essential for MDMX functions: (1) MDMX/p53 interaction inhibits p53 DNA binding. (2) MDMX/MDM2 interaction promotes MDMX degradation. (3) MDMX/14-3-3 interaction promotes MDMX nuclear translocation. (4) MDMX/CK1α interaction promotes MDMX/p53 binding and inhibition of p53. (5) MDMX intramolecular interaction regulates p53 binding. Importantly, these interactions are regulated by two phosphorylation sites (S289, S367) located in intrinsically disordered regions (IDR) of MDMX. We hypothesize that the coordinated control is mediated by intramolecular interactions between ordered and disordered regions of MDMX. The IDRs regulate the switching of intra/inter-molecular contacts in a phosphorylation-dependent manner. We propose integrated molecular biology and biophysics approach to elucidate the mechanism of MDMX regulation: (1) Determine how the length and flexibility of MDMX IDR1 regulates auto- inhibition of p53 binding. (2) Investigate how CK1α phosphorylation switches MDMX to an open conformation that inhibits p53 DNA binding. (3) Determine how Chk2 phosphorylation of IDR2 S367 regulates multiple MDMX interactions. These experiments will significantly advance the understanding of how stress signals activate p53 by targeting disordered regions of MDMX, which is essential to develop MDMX inhibitors for cancer therapy.