The disordered region 1 of Mdm2 weakens p53-binding in both its unmodified and hyperphospho-forms

Mdm2, the main negative regulator of the "tumor-suppressor" p53, is an attractive target for anti-cancer treatments, but the molecular mechanisms of its action are still elusive. Indeed, producing purified samples for its structural analysis is difficult: Mdm2 is a multidomain protein, alternating folded and disordered regions, the latter being moreover natively hyperphosphorylated. Here, we report the structural study of Mdm2 constructs including its folded p53-binding domain (p53-BD) and Zinc-Finger, and their spacer, a 190-residue long disordered region (IDR1) hyperphosphorylated by CK1. Using NMR spectroscopy, we revealed that IDR1 establish ultraweak intramolecular contacts with p53-BD in the μs timescale, which provokes the partial release of the N-terminal tail from the p53-binding groove of Mdm2. Then, using ITC, we established that these Mdm2 intramolecular interactions decrease the affinity for p53 by up to 25-fold compared to the isolated p53-binding domain. Surprisingly, the identified 15 CK1-established phosphosites between Mdm2-S192 and -S286 do not improve the affinity for p53 despite their binding to the positively charged DNA-binding domain. Structural studies on multidomain proteins are scarce, and even more rare on natively phosphorylated species. This report shows how disordered regions can interfere with folded domains, and markedly affect binding between cognate cellular partners.