C-Terminal Substitution of MDM2 Interacting Peptides Modulates Binding Affinity by Distinctive Mechanisms

The complex between the proteins MDM2 and p53 is a promising drug target for cancer therapy. The residues 19–26 of p53 have been biochemically and structurally demonstrated to be a most critical region to maintain the association of MDM2 and p53. Variation of the amino acid sequence in this range obviously alters the binding affinity. Surprisingly, suitable substitutions contiguous to this region of the p53 peptides can yield tightly binding peptides. The peptide variants may differ by a single residue that vary little in their structural conformations and yet are characterized by large differences in their binding affinities. In this study a systematic analysis into the role of single C-terminal mutations of a 12 residue fragment of the p53 transactivation domain (TD) and an equivalent phage optimized peptide (12/1) were undertaken to elucidate their mechanistic and thermodynamic differences in interacting with the N-terminal of MDM2. The experimental results together with atomistically detailed dynamics simulations provide insight into the principles that govern peptide design protocols with regard to protein-protein interactions and peptidomimetic design

Improved eIF4E Binding Peptides by Phage Display Guided Design: Plasticity of Interacting Surfaces Yield Collective Effects

10.1371/journal.pone.0047235PLoS ONE710

Structural comparison of N-Capping motifs either containing S or D at position 5 in eIF4E interacting peptides when bound to eIF4E. A)

<p>Crystal structure of the eIF4G1-D5S derivative peptide bound to eIF4E. It can be seen that S5 makes two optimized hydrogen bonds to the amide backbone groups of R6 and E7 located in the first turn of the helix of the bound peptide. The electron density for the eIF4G1-D5S peptide in the 2Fo-Fc map is shown with the blue mesh and is contoured at 1.5σ. <b>B)</b> D5 in the 2W97 structure bound to the eIF4G1 wild type peptide also makes these two hydrogen bonds but their geometry is not as optimal as that seen for the hydrogen bonds formed in the eIF4G1_D5S peptide.</p

Calculated K_ds and derived ΔG° (Gibbs free energy of binding) for the interactions between eIF4E and the N-Cap derivative peptides.

<p>The table shows the peptide sequences used to study the influence of alternative residues and their effects in capping the first turn of the α-helix when bound to eIF4E. K_ds were determined using SPR with eIF4E immobilized on the chip surface. K_ds were derived from the equilibrium responses (K_eq) and from the association and dissociation phases (K_kin) of the SPR data. The Gibbs free energy of binding (<b>ΔG°</b>) was calculated with the equation ΔG = –RT ln K_a using both dissociation constant values determined.</p

Q8 stabilizes the N-terminal end of the α-helix in eIF4E interacting peptides and facilitates the librational movement of the PHAGESOL peptide across the surface of eIF4E.

<p>A) A representative snapshot showing the formation of an intra-molecular hydrogen bond between the side chain of Q8 and the backbone amide of S5. The formation of this hydrogen bond stabilizes the N-terminal portion of the helix in the bound peptide. The snapshot was taken from the simulation for the KKRYSRDQLLAL peptide bound to eIF4E. B) Overlay of representative snapshots from the PHAGESOL (KKRYSRDQLVAL) and eIF4G1-G11A (KKRYDREFLLAF) peptide simulations when bound to eIF4E. V10 of the PHAGESOL (orange) peptide orients itself and the helical turn it is located on, into a position where it can occupy the volume of space that would otherwise be occupied by the conserved L as shown here on the eIF4G1 derivative peptide (magenta). The movement of V10 across the surface of eIF4E results in the helical segment of the peptide spatially re-orientating itself in contrast to the other derivative peptides. This conformational change is facilitated by the presence of Q8 that stabilizes the N-terminal helix of the peptide and forms few interactions with eIF4E in contrast to F8 in eIF4G1-G11A. Deviations in the planarity of the tyrosine and phenylalanine ring systems are within the tolerances of the torsional restraints of the MD simulations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047235#pone.0047235-Macias1" target="_blank">[31]</a>.</p

Comparison of 4EBP1 and eIF4G1 peptides suggests that eIF4E interacting peptides can form an ensemble of conformations when in complex with eIF4E.

<p><b>A)</b> An overlay of two eIF4E crystal structures complexed with either a 4EBP1 (1EJ4) or eIF4G1 (2W97) derived peptide demonstrating the deviation in their C-terminal structural conformations. 4EBP1 is shown in salmon and eIF4G1 in cyan. <b>B)</b> A plot of the φ and ψ angle distribution, derived from the 50 ns simulations of the peptides eIF4G1 and 4EBP bound to eIF4E, for the residues L10 and M10 respectively. <b>C)</b> A plot of the φ and ψ angle distributions, derived from the 50 ns simulations of peptides eIF4G1 and 4EBP bound to eIF4E, for the residues G11 and E11 respectively. <b>D)</b> A plot showing the distribution of distances, for the peptides eIF4G1 and 4E-BP1 when bound to eIF4E, between the Cα atoms of residues 6 and 10 versus the distance between the Cα atoms of residues 8 and 12. The distances were calculated from their respective 50 ns simulations for both peptides. <b>E)</b> A histogram of the angular distribution between the Cα atoms of positions 6, 8 and 10 of the eIF4G1 and 4EBP1 peptides from the 50ns simulations respectively.</p

Data collection and refinement statistics.

<p>Crystallographic data and refinement statistics for eIF4E in complex with m⁷GTP and eIF4G1-D5S (PDB ID: 4AZA).</p

The precise sites of interaction for eIF4E interacting peptides are non-identical and display distinctive conformational differences.

<p><b>A)</b> Overlay of representative snapshots from the VL-PHAGESOL-Q8F (cyan) and VL-PHAGESOL (salmon) peptide simulations bound to eIF4E. The deviation in helical movement between these two peptides is dramatic with F8 in the VL-PHAGESOL-Q8F peptide forming extensive interactions with eIF4E and preventing the lateral movement across the surface of eIF4E seen for VL-PHAGESOL. The lateral movement of VL-PHAGESOL is facilitated by Q8, which forms a hydrogen bond with S5 of the bound peptide, and also forms less interactions with eIF4E. It also allows the C-terminal of the helix to unwind and allow L12 to pack optimally into the volume that F8 would occupy in the other peptide. <b>B)</b> Overlay of representative snapshots from the PHAGESOL and VL-PHAGESOL peptide simulations bound to eIF4E. These peptides only differ at the position 10 with PHAGESOL containing a V and VL-PHAGESOL possessing an L. The presence of Q8 allows the helix to move more independently enabling the residue at position to dictate the final packing arrangements of the peptide. V10 packs optimally in the PHAGESOL peptide against the surface of eIF4E which induces the lateral movement of the peptide. L12 can still pack optimally. L10 in the VL-PHAGESOL peptide also packs against a hydrophobic area of eIF4E, closely located to where V10 packs, but due to the greater length of the alkyl chain allows the peptide to pack in a different conformation with eIF4E. A conformation where the C-terminal end unwinds to allow L12 to pack into an alternative optimal position against eIF4E. <b>C)</b> and <b>D)</b> are plots showing the distribution of the relative positions of the helical portion of the eIF4E bound peptide with respect to the surface of the protein throughout their individual simulations. The plots show the distinct conformational differences of the peptides in their interactions with eIF4E. The PHAGESOL peptide (magenta) and VL-PHAGESOL (yellow) both have very distinct conformational populations whilst the VL-PHAGESOL-Q8F/L12F peptide (red) has a much more dispersed population that overlaps with the conformational space of the VL-PHAGESOL peptide (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047235#pone-0047235-g004" target="_blank">figure 4S</a> for definition of the conformational measurement made). Deviations in the planarity of the tyrosine and phenylalanine ring systems are within the tolerances of the torsional restraints of the MD simulations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047235#pone.0047235-Macias1" target="_blank">[31]</a>.</p

Detrimental interactions that attenuate peptide binding to eIF4E when D or E are incorporated at position 5. A)

<p>Snapshot from the 50 ns computer simulation of the eIF4G1 wild-type peptide bound to eIF4E. In the eIF4G1 wild type peptide the D5 side chain is able to hydrogen bond with R186 of the receptor, deforming the N-terminal helix of the bound peptide. This deformation causes E7 of the peptide to interact more frequently with R6, which is also found on the peptide. This interaction disrupts the electrostatic interaction between R196 found on the surface of eIF4E and E7 of the eIF4G1 peptide, which helps to stabilize the formation of the peptide:protein complex. <b>B)</b> The average distance between E7 and R6 throughout the 50 ns simulations for the bound peptides eIF4G1-D5S, eIF4G1-T5S and the wild-type peptide. The average distance frequently dips below 3.2 Å for the wild type peptide compared to the S5 or T5 derivative peptides, which indicates that E7 and R6 interact more frequently with each other and destabilize complex formation in the wild type peptide compared to the derivative peptides. <b>C)</b> A snapshot from the 50 ns simulation of the complex between eIF4G1-D5E and eIF4E, showing the formation of a loop-like structure preceding the N-terminus of the peptide. The formation of the loop structure arises from the electrostatic interaction of E5 of the peptide with K2 at the N-terminus of the peptide. <b>D)</b> The distances between the two O atoms (the red and blue lines on the plot) of the E5 side chain and the N atom of the K1 side chain were plotted over the course of the simulation. The plot reveals that for a significant portion of the simulation these residues are within 3.2 Å of each other indicating the formation of a stable electrostatic interaction. This interaction hinders the interaction of K1 with E132 on the surface of eIF4E and leads to further destabilization of the eIF4E-peptide complex. Deviations in the planarity of the tyrosine and phenylalanine ring systems are within the tolerances of the torsional restraints of the MD simulations. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047235#pone.0047235-Macias1" target="_blank">[31]</a>.</p

Calculated K_ds and derived ΔG° (Gibbs free energy of binding) for the interactions between eIF4E and the hybrid eIF4G1/PHAGESOL peptides.

<p>The table shows the peptide sequences used to study the relevance of individual amino acid changes observed in the phage derived sequence. The peptides were characterized using a fluorescence based thermal denaturation method and by using SPR with eIF4E amine coupled to the chip surface. K_ds were also derived from the respective thermal shift and SPR data. K_ds were derived from the equilibrium responses (K_eq) and from the association and dissociation phases (K_kin) of the SPR data. The Gibbs free energy of binding (<b>ΔG°</b>) was calculated with the equation ΔG = −RT ln K_a using both dissociation constant values determined.</p