Interleukin-25 recognition by its unique receptor IL-17Rb via two discrete linear and cyclic epitopes.

Interleukin-17 (IL-17) is a family of pro-inflammatory cytokines and has been involved in the pathogenesis of chronic inflammatory and autoimmune diseases. The IL-17E, also known as IL-25, is a distinct member of this family that binds to its unique receptor IL-17Rb to induce the activation of nuclear factor kappa-light-chain enhancer of activated B cells. Here, we systematically examined the intermolecular recognition and association of IL-25 with IL-17Rb and demonstrated that the IL-25 primarily adopts two discrete linear and cyclic epitopes to interact with IL-17Rb. The two epitopes are separately located in the monomers 1 and 2 of IL-25 homodimer and cover sequences 125 DPRGNSELLYHN136 and 77 ELDRDLNRLPQDLY90 . They totally contribute 71.6% binding energy to the full-length IL-25. The linear epitope targets a site spanning over the extracellular fnIIID1 and fnIIID2 domains of IL-17Rb, while the cyclic epitope primarily binds at the fnIIID1 domain. In addition, we also found that the linear and cyclic epitopes are natively folded into ordered single-stranded and double-stranded conformations in IL-25 protein context, respectively, but would become largely disordered when splitting from the context to be free peptides, which, however, cannot bind effectively to IL-17Rb as them in the native state. In this respect, we extended the cyclic epitope to cover the whole IL-25 double-stranded region and added a disulfide bridge across its two strands at three selected anchor residue pairs. It is revealed that the disulfide-stapled peptides can be constrained into a native-like conformation and thus exhibit an improved binding potency to IL-17Rb as compared to their unstapled counterpart.