The Distribution and Occupancy of Specific Lid-Receptor Interactions.

<p>The distribution of the high occupancy contacts and salt bridges obtained from MD simulations of the phosphorylated MDM2-pS17 and phosphomimetic MDM2-S17D lid. The occupancies for pS17 are shown in blue filled bars and S17D in red filled bars. The analysis is focused on the dynamics of specific interactions formed by the lid with the core domain of MDM2. The intra-domain salt bridges are not included. The distance cutoff for hydrogen bonding is 3.50 Å and the angle cutoff is 120.00 degrees.</p

Mutation-induced Modulation of the MDM2 Conformational Ensembles: A Truncated Lid Model.

<p>Structural clustering of MD trajectories from simulations with a truncated lid (residues 16–24). The effect of phosphorylation and mutation-induced modulation of the conformational ensembles is illustrated for MDM2-WT (<b>A</b>), MDM2-pS17 (<b>B</b>) and MDM2-S17D (<b>C</b>). The apo-MDM2 (16–109) construct was used in all simulations. The representative MDM2 conformations from 10 dominant clusters were subjected to subsequent structural refinement by global minimization of the complete MDM2 structure. A ribbon-based representation of the MDM2 conformational ensembles was used. Coloring is according to the B-factors values (blue-to-red spectrum) reflecting protein nobilities of the MDM2 residues (from more rigid-blue regions to more flexible-red regions).</p

The Lid-based Modulation of Conformational Plasticity in the MDM2 Receptor: Analysis of Solvent Accessible Surface Area Changes.

<p>Change in the percentage of the accessible surface area (ASA) of the receptor residues upon binding of the phosphorylated lid in 3 lowest energy clusters from docking simulations (<b>A</b>), (<b>B</b>) and (<b>C</b>). (<b>D</b>) The change in the percentage of ASA for the receptor side-chains averaged over 10 lowest energy clusters from docking simulations. The crystal structure of the p53-MDM2 complex was used as the reference structure. Positive (negative) values indicate that the residues get more exposed (buried) upon binding of the lid as compared to their position in the crystal structure of the p53-MDM2 complex.</p

Mutation-induced Modulation of the MDM2 Conformational Ensembles: A Complete Lid Model.

<p>Structural clustering of MD trajectories from simulations with a complete lid (residues 1–24). The effect of phosphorylation and mutation-induced modulation of the conformational ensembles is illustrated for MDM2-WT (<b>A</b>), MDM2-pS17 (<b>B</b>) and MDM2-S17D (<b>C</b>). The complete apo-MDM2 (1–109) construct was used in simulations. The representative lid conformations are colored in red (residues 1–15) and cyan (residues 16–24). A ribbon-based representation of the MDM2 core domain was used.</p

Molecular mimicry of the p53-MDM2 interactions in the predicted structure of the phosphorylated pS17 lid.^*

*<p>The table presents the residues and the corresponding atoms of MDM2 receptor which are involved in hydrogen bond formation with p53 residues and the phosphorylated lid residues.</p

Models of the MDM2-mediated Regulation Mechanisms.

<p>(<b>A</b>) The first model assumed a conformational change in the N-terminus amphipathic helix of p53 upon phosphorylation of T18. According to this model, conformational changes in p53 could reduce binding affinity and result in the eventual disruption of the p53-MDM2 interactions. (<b>B</b>) The second model suggested the role of phosphorylation at S17 in displacing the phosphorylated p53 from the binding site. A simultaneous in-vivo phosphorylation of T18 and S20 on p53 and S17 on the MDM2 lid can bring the negatively charged phosphates on these residues in a close proximity leading to the abrogation of the p53-MDM2 interactions.</p

The Interaction Network of the Predicted Closed Form for the Phosphorylated pS17 Lid Interactions with MDM2.

<p>(<b>A</b>) The “anchoring” interactions of the phosphorylated lid formed by E23, Q24 and E25 residues K51 and Y100 core domain residues. E23 and E25 are shown in red sticks, K51 in blue sticks, Q24 and Y100 in light blue sticks. (<b>B</b>) The hydrogen bonds formed by pS17 and Q18 with K94, Q72 and H73 residues. PS17 is shown in ball-stick-model with atom-based coloring, K94 in blue sticks. (<b>C</b>) These interactions were further supported by the favorable contacts of P20 occupying the first hydrophobic pocket and A21 backbone with the H96 and R97 residues. The hydrogen bonding network is supported by the packing interactions of the lid with the L54, H96 and Y100 residues. (<b>D</b>) The MDM2 residues L54, L57, G58, I61, M62, V93, H96 and I99 are involved in the hydrophobic contacts of both p53 and pS17 lid. I19 is not projected towards the binding cleft; because of it restricted movement due to P20.</p

Folding-binding Coupling of the Phosphorylated pS17Lid.

<p>(<b>A</b>) The initial “open “conformational state of the lid shown over the p53 binding surface of MDM2 receptor. The three hydrophobic pockets (I, II, III) the receptor are occupied by respective p53 residues L26, W23 and F19 respectively in the p53-MDM2 complex. (<b>B</b>) The “closed” conformational state of the lid. Conformational changes in the MDM2 receptor and the phosphorylated lid upon binding are depicted. Structural restructuring and partial unfolding of the structured turn in the lid upon binding is highlighted in magenta. Positions of P20 and I19 on the MDM2 surface and phosphorylated S17 near K94 and H73 are also shown. Residues around the p53-binding site which undergo large conformational change upon lid binding are highlighted.</p

The Lid-based Modulation of Conformational Plasticity in the MDM2 Receptor: Analysis of Side-Chain Movements.

<p>Side chain movements in the MDM2 receptor upon binding of the phosphorylated pS17 lid in 3 lowest energy clusters from docking simulations are shown in (<b>A</b>)<b>,</b> (<b>B</b>) <b>and</b> (<b>C</b>)<b>.</b> (<b>D</b>) The RMSD values of the receptor side-chains averaged over 10 lowest energy clusters from docking simulations. The RMSD of the receptor side-chains were calculated using the crystal structure of the p53-MDM2 complex as the reference state.</p

MD Simulations of the Phosphorylated pS17 MDM2 form.

<p>MD simulations were carried out using a truncated lid form (residues 16–24). The RMSD fluctuations of the Cα atoms of the core MDM2 domain residues 25–109 (<b>A</b>) and the MDM2 lid residues 16–24 (<b>B</b>) obtained from 10 ns MD simulations of the phosphorylated pS17 form. (<b>C</b>) The RMSF values of the Cα atoms of the core MDM2 domain residues 25–109 (shown in red) and the MDM2 lid residues 16–24 (shown in blue) obtained from 10 ns MD simulations of the phosphorylated pS17 form.</p