The Effects of Phosphomimetic Lid Mutation on the Thermostability of the N-terminal Domain of MDM2

Chemical states of the N-terminal “lid” of MDM2 regulate p53 binding: Simulations reveal complexities of modulation

“…6 and 7); by 17 ns, R97 also makes an interaction with the phosphate. This latter observation seems to be in accord with the experimental findings of Worrell et al 13 To get an estimate of the strength of the interaction between the lid and the cleft upon phosphorylation (at S17), the -2e charge on the phosphate is switched off, and we find a 110 kcal/mol destabilization in the electrostatic energy and a further loss of ~286 kcal/mol of solvation energy, thus highlighting the strong electrostatic interactions between the phosphate and the MDM2 surface. Our results now build upon experimental observations, 8,11 demonstrating that the lid is very flexible and not stabilized in any specific location on the cleft.…”

“…2) although the gatekeeper Y100, 4 remains in the closed conformation; nevertheless the binding site is open enough for initial encounter complexes to form. 19,20 This now provides a molecular picture behind the experimental observations 11,13 whereby the presence of the lid can occlude MDM2 from binding p53 and yet allow small molecules are apparent from our simulations, a feature that has also been reported earlier 20 ). WTc showed a transition of the lid from a closed state to an open state during the simulation, suggesting a very weak association; however the transition from open to closed state was not observed for either WTc and WTo (see Movie M1a-d) the timescale of this equilibrium exists in the millisecond regime.…”

“…Consistent with this, phospho-mimetic lid mutation also increases the thermostability of the N-terminal domain of MDM2 in the presence of Nutlin or p53-mimetic peptides. 13 This is in contrast to the NMR derived hypothesis that the ©2 0 1 1 L a n d e s B i o s c i e n c e .…”

A Novel p53 Phosphorylation Site within the MDM2 Ubiquitination Signal

“…Phosphomimetic mutation of these sites can stimulate p53-dependent transcription (2), and mouse transgenes with alanine-substituted mutations have increased cancer incidence in stress or tissue-specific manner (3)(4)(5). The biochemical basis for these effects has been reported; transactivation domain phosphorylation at Thr 18 can directly inhibit MDM2 binding (6,7), whereas Ser 20 phosphorylation can stabilize p300 binding (8,9). Ser 392 phosphorylation can stimulate p53 sequence-specific DNA binding according to the ensemble model of allostery (10) in part by stabilizing p53 tetramers from the dimeric state (11).…”

“…MDM2-mediated degradation of p53 is the best characterized of the p53-inhibiting pathways (14) that can in turn be stimulated by MDM2 interactions with other proteins, including TAFII250 (15) and MDMX (16). Although there are many phosphoacceptor sites on MDM2 that have been identified, the best characterized site with potential to stimulate MDM2-mediated inhi-bition of p53 is in the N-terminal MDM2 pseudosubstrate motif, or "lid" (17,18). In addition to the MDM2-stimulated degradation pathway, there are three distinct kinase pathways that directly target p53 and inhibit the protein function, although the molecular basis for this inhibition is not known.…”