Figure 1.
Sequence alignment of peptide ligands targeting the Mdm2 N-terminal domain.
The critical interacting residues (F19, W23 and L26) in the p53 wild-type peptide and conserved in the indicated stapled peptides are shaded. The staple tethering site is denoted by ‘X’ and chemical structure of the staple moiety (adapted from [18]) is shown below.
Figure 2.
Crystallographic Unique Complexes of the M06 stapled peptide bound to the N-terminal p53 binding domain of Mdm2-M62A (6–125).
The 2Fo-Fc electron density map, which has been contoured at 1.2 σ, clearly demonstrates the presence of the whole peptide bound to Mdm2.
Figure 3.
Mdm2-M62A (6–125) shows a high degree of conformational plasticity in the α2’ and ‘hinge’ helices capping the p53-binding pocket.
A,B The binding pockets that form when Mdm2 interacts with SAH-8 (A) and p53 WT peptide (B) are much more open when compared to the binding pockets formed in the two complexes of Mdm2:M06 found in the asymmetric unit. The key residue that is involved in forming the bottom of the binding pocket in the Mdm2 complexes with SAH-8 (A) and p53 WT (B) is Y100 (shown in red) on the α2’ helix, where it either closes the binding site by orientating itself inward or out towards the solvent, respectively. C, D In contrast, the binding pockets formed in the two crystallographically unique M06 complexes are capped by the ‘hinge’ helix of the lid (residues 20–24, highlighted in yellow). The helix is stabilized in this conformation by a hydrogen bond between Q24 (magenta) and the amide cap at the C-terminal of M06.
Table 1.
Crystallographic data collection and refinement statistics.
Figure 4.
Comparison of Mdm2-M62A:M06 and Mdm2:SAH-8 structures.
A, The staple in both Mdm2:M06 complexes, found in the asymmetric unit, packs against residues L54, F55 and more closely with G58 (shown in red) in the absence of M62. B, The conformation of the staple in SAH-8 differs subtly from that seen in the M06 complexes, with the staple in SAH-8 deformed by the interaction with M62 (shown in blue). C, The piperazinone ring of Nutlin forms critical contacts with M62, which are lost in the Nutlin resistant Mdm2. D, Overlay of the Mdm2-M62A:M06 complex with the Mdm2:SAH-8 complex, highlighting the conformational differences between the respective hydrocarbon staples in the presence and absence of M62. M06 is highlighted in yellow and SAH-8 in orange. The conformation of the F55 side-chain also changes, but this is primarily due to crystal packing effects.
Figure 5.
Differential orientation of Y100 in the Mdm2-M62A:M06 structure.
A, Overlay of the two Mdm2:M06 complexes from the asymmetric unit of the crystal structure showing the packing interactions at the bottom of the binding pocket. In both complexes the ‘hinge’ helix caps the bottom of the pocket and is stabilized via a hydrogen bond between Q24 and M06. However, in the M06 complex highlighted in blue, the ’hinge’ helix swings further into the binding pocket and results in I19 preventing Y100 from orientating itself in towards the binding pocket. In contrast, the degree to which the helix moves into the pocket is less in the other M06 complex (shown in red). This results in Y100 forming a hydrogen bond to L26 and I19 packing alongside it to form the bottom of the binding pocket. B, In the Mdm2:Nutlin complex the ‘hinge’ helix caps the bottom of the binding pocket as seen in A. However, the Y100 adopts an intermediate position between the two orientations observed in the M06:Mdm2 complexes, with the I19 packing on top of it. In addition, H96 interacts with the chloro-benzyl moiety of Nutlin, causing the helix to swing into the peptide binding groove, inducing the pocket to narrow. C, Capping of the Mdm2 binding pocket by the ‘hinge’ helix does not occur for the SAH-8 complex due to steric hindrance. This is mainly due to the SAH-8 stapled peptide possessing a 2 amino acid extension compared to M06.
Figure 6.
Flexibility of Y100 seen in Molecular Dynamics simulations of Mdm2.
Molecular dynamics simulations (100 ns each) of Mdm2 in its apo, Nutlin-bound, p53 peptide-bound and stapled-peptide bound states shows that Y100 accesses 3 different conformational substrates as measured by the distribution of its chi1 torsional angle: a closed state that stabilizes Nutlin-like molecules, a fully open state that is seen in wild type and in the destabilized Nutlin-M62A complex, and an open state that stabilizes the peptides. Simulations were carried out as previously described [19].