Abstract
Diversity and complexity of MDM2 mechanisms govern its principal function as the cellular antagonist of the p53 tumor suppressor. Structural and biophysical studies have demonstrated that MDM2 binding could be regulated by the dynamics of a pseudo-substrate lid motif. However, these experiments and subsequent computational studies have produced conflicting mechanistic models of MDM2 function and dynamics. We propose a unifying conformational selection model that can reconcile experimental findings and reveal a fundamental role of the lid as a dynamic regulator of MDM2-mediated binding. In this work, structure, dynamics and energetics of apo-MDM2 are studied as a function of posttranslational modifications and length of the lid. We found that the dynamic equilibrium between “closed” and “semi-closed” lid forms may be a fundamental characteristic of MDM2 regulatory interactions, which can be modulated by phosphorylation, phosphomimetic mutation as well as by the lid size. Our results revealed that these factors may regulate p53-MDM2 binding by fine-tuning the thermodynamic equilibrium between preexisting conformational states of apo-MDM2. In agreement with NMR studies, the effect of phosphorylation on MDM2 interactions was more pronounced with the truncated lid variant that favored the thermodynamically dominant closed form. The phosphomimetic mutation S17D may alter the lid dynamics by shifting the thermodynamic equilibrium towards the ensemble of “semi-closed” conformations. The dominant “semi-closed” lid form and weakened dependence on the phosphorylation seen in simulations with the complete lid can provide a rationale for binding of small p53-based mimetics and inhibitors without a direct competition with the lid dynamics. The results suggested that a conformational selection model of preexisting MDM2 states may provide a robust theoretical framework for understanding MDM2 dynamics. Probing biological functions and mechanisms of MDM2 regulation would require further integration of computational and experimental studies and may help to guide drug design of novel anti-cancer therapeutics.
Highlights
The p53 tumor suppressor is known as ‘‘The Guardian of the Genome’’ [1] and plays a fundamental role in maintaining the integrity of the genome by inducing either cell cycle arrest or apoptosis following cellular stress signals [2,3]
Despite important differences in functional dynamics of apo-MDM2 as a function of lid length and composition (Figure 5), we found that the dynamic equilibrium between ‘‘closed’’, and ‘‘semi-closed’’ lid forms may be a fundamental characteristic of MDM2 regulatory interactions, which can be modulated by phosphorylation, phosphomimetic mutation as well as by the lid size
We determined that the dynamic equilibrium between ‘‘closed’’, and ‘‘semi-closed’’ lid forms may be an important characteristic of MDM2 regulatory interactions, which can be modulated by phosphorylation, phosphomimetic mutation as well as by the lid size
Summary
The p53 tumor suppressor is known as ‘‘The Guardian of the Genome’’ [1] and plays a fundamental role in maintaining the integrity of the genome by inducing either cell cycle arrest or apoptosis following cellular stress signals [2,3]. In normal cells the level of p53 is tightly regulated and maintained at a low level by the murine double minute (MDM2) oncoprotein, which is a p53specific E3 ubiquitin ligase [4,5,6]. MDM2 recognizes the Nterminal trans-activation domain of the p53 tumor suppressor and is responsible for inactivation of p53 transcription and targeting p53 for ubiquitin-mediated degradation [7,8,9]. MDM2 can inhibit p53 activity by nucleo-cytoplasmic shuttling [10,11] or by binding of its N-terminal domain to the trans-activation domain of p53 [12,13]. MDM2 consists of several conserved domains including the N-terminal domain that binds the a-helix from the N- terminal transactivation domain of p53 [10,11,12].
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