Interleukin-1α, interleukin-1β and interleukin-1 receptor antagonist share a common U-shaped recognition epitope on interleukin-1 receptor surface.

Interleukin-1 (IL-1) plays a central role in the regulation of immune and inflammatory responses. There are two forms of IL-1 agonists (IL-1α and IL-1β) and one form of IL-1 antagonist (IL-1Ra); they share a similar binding mode to the IL-1 receptor (IL-1R) but exhibit opposite biological functions on the receptor. In this study, the intermolecular interactions of IL-1R receptors with IL-1α, IL-1β and IL-1Ra ligands were systematically investigated at structural, energetic and dynamic levels. It was found that the receptor primarily adopts a U-shaped, double-stranded and linear/conformational-hybrid epitope to commonly interact with the three ligands. The epitope covers a common protein segment (residues 107-127), which is fully located within the C2T2 subdomain of the IL-1R extracellular domain and contributes ~40% to the total binding energy of IL-1R/ligand association. The epitope is natively folded into an ordered conformation in the IL-1R protein context but would become largely disordered out of the context. Here, we adopted a disulfide bridge to staple U-shaped epitope-derived peptides, which can be effectively constrained into a native-like conformation and thus exhibit an improved affinity to ligands as compared to their unstapled counterpart, with affinity increase by up to ~15-fold. These disulfide bridges were designed to point out of ligand/peptide complex interface and thus would not disrupt the direct complex interaction. Energetic decomposition imparted that the stapling has only a modest influence on the interaction enthalpy and desolvation effect of ligand/peptide binding, but can substantially reduce entropy penalty upon the binding. For a peptide, the stapling-addressed entropic reduction can be roughly regarded as a constant, which only improves peptide affinity to these ligands, but does not change peptide selectivity over different ligands.