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Abstract

As primary p53 antagonists, Mdm2 and the closely related Mdm4 are relevant cancer therapeutic targets. We have previously described a series of cell-permeable stapled peptides that bind to Mdm2 with high affinity, resulting in activation of the p53 tumour suppressor. Within this series, highest affinity was obtained by modification of an obligate tryptophan residue to the non-natural L-6-chlorotryptophan. To understand the structural basis for improved affinity we have solved the crystal structure of this stapled peptide (M011) bound to Mdm2 (residues 6–125) at 1.66 Å resolution. Surprisingly, near identity to the structure of a related peptide (M06) without the 6-chloro modification is observed. Further analysis of linear and stapled peptides comprising 6-Me-tryptophan provides mechanistic insight into dual Mdm2/Mdm4 antagonism and confirms L98 of Mdm4 as a mutable steric gate. The results also highlight a possible role of the flexible hinge region in determining Mdm2/Mdm4 plasticity.

Structural determination of M011 bound to Mdm2-M62A

The sequence compositions of peptides used in this study are shown in . The crystal structure of M011 bound to Mdm2-M62A shows 2 unique complexes in the asymmetric unit (, ). Within each, a single molecule of M011 interacts with a pronounced hydrophobic cleft that binds endogenous p53 (residues 19 to 26). As with other peptide antagonists, an interfacing signature triad of F19, W23 (6-chloro) and L26 residues (numbering based on p53 sequence) project their side chains into discrete apolar sub-pockets (“Phe”, “Trp” and “Leu” pockets) (). Comparison with the structure of a similar stapled peptide (M06) [], comprising native tryptophan bound to the same Mdm2 construct shows remarkable similarity. Each M011 complex, from the asymmetric unit, shows high structural similarity to the corresponding complex bound to M06 (all-atom derived RMSDs = 0.167 and 0.126 Å). This is particularly evident in the binding site, where minimal variation is observed in the ligand conformations (all-atom RMSDs = 0.055 and 0.031 Å). The major difference arises from rotation around the CHI1 bond of the L57 side chain in Mdm2 to widen the Trp pocket, with other residues comprising this pocket (F86, F91, I99) remaining unchanged (). This optimal formation of Van der Waals contacts around the 6-chloro moiety, resulting from the change in conformation of L59, explains the ~8-fold increased binding affinity of M011 over the parental stapled peptide lacking the 6-chloro modification (PM2, ). Structural differences are also observed in the lid/hinge region between the two M011: Mdm2 complexes in the asymmetric unit (). These result in a small conformational change of the M011 L26 residue. The observed changes in the hinge regions are primarily the result of crystal packing effects. However, these differences highlight how capping of the peptide binding groove by the hinge region can potentially impact on the binding of ligands to Mdm2.