Exploring the effect of Hydrocarbon Cross-linkers on the Structure and Binding of Stapled p53 Peptides

Short-length peptides are used as therapeutics due to their high target specificity and low toxicity, for example, peptides designed for targeting the interaction between oncogenic protein p53 and E3 ubiquitin ligase MDM2. These peptide therapeutics form a class of successful inhibitors. To design such peptide-based inhibitors, stapling is one of the methods in which amino acid side chains are stitched together to get conformationally rigid peptides, ensuring effective binding to their partners. In the current work, we use computer simulations to investigate p53 peptides stapled with hydrocarbon chains of different lengths and positions of attachment to the peptide. We subsequently analyze their binding efficiency with MDM2. The introduction of stapling agents restricts the conformational dynamics of peptides, resulting in higher persistence of helicity. The efficiency of the stapling agents has also been verified imposing these stapled peptides to adverse conditions viz. thermal and chemical denaturation. In addition, the conformational exploration of peptides has been investigated using Temperature replica exchange molecular dynamics (T-REMD) simulations. From both the unbiased and T-REMD simulations, p53 with a long hydrocarbon cross-linker shows a more conformationally rigid structure having high helicity compared to other stapled peptides. The rigidity gained due to cross-linking reduces the entropy of the peptide in the free state and thereby facilitates the complexation process. From the binding studies, we have shown that the peptide having multiple short staples has shown a larger enthalpy change during binding free energy, resulting from its orientation and interactions of residues in the binding interface. On the other hand, a peptide with a single long stapling agent shows less entropic penalty than other systems. Our studies suggest a plausible rationale for the relation between the length and the position of attachment of cross-linkers to peptides and the binding free energies between the peptides and their target partners.