Introducing the Potential Binding Interface between the TRAIL-Mimicking Peptide and DR5 via Alanine Scan

Here we harnessed the unexplored binding interface between the 16-residue peptide (P) agonist and death receptor 5 (DR5). P is a solitary peptide ligand that mimics TRAIL (the natural ligand to death receptor) and is reported to control cancer growth <i>in vivo</i> selectively. We delved into the strategic merging of experimental and <i>in silico</i> structure-activity studies via the alanine scanning mutagenesis of P, wherein the disulfide bond was kept intact for structural integrity. Antiproliferative activity studies with these synthetic mutants on HCT116 cells enabled the mapping of the interaction engagement of each residue. Further, <i>in silico</i> docking and MD simulations led us to interpret and model the 3D interface of the binding site. Notably, Trp1, Leu4, Arg7, Ile8, Gln12, and Arg15 were projected experimentally as "hot-spot" residues crucial for primary interactions with DR5, which is predominantly supported via <i>in silico</i> investigations. This study is pivotal for developing new-generation peptide agonists that induce death receptor-mediated apoptosis.