Abstract

Abstract Abiotic d -proteins that selectively bind to natural l -proteins have gained significant biotechnological interest. However, the underlying structural principles governing such heterochiral protein–protein interactions remain largely unknown. In this study, we present the de novo design of d -proteins consisting of 50–65 residues, aiming to target specific surface regions of l -proteins or l -peptides. Our designer d -protein binders exhibit nanomolar affinity toward an artificial l -peptide, as well as two naturally occurring proteins of therapeutic significance: the D5 domain of human tropomyosin receptor kinase A (TrkA) and human interleukin-6 (IL-6). Notably, these d -protein binders demonstrate high enantiomeric specificity and target specificity. In cell-based experiments, designer d -protein binders effectively inhibited the downstream signaling of TrkA and IL-6 with high potency. Moreover, these binders exhibited remarkable thermal stability and resistance to protease degradation. Crystal structure of the designed heterochiral d -protein– l -peptide complex, obtained at a resolution of 2.0 Å, closely resembled the design model, indicating that the computational method employed is highly accurate. Furthermore, the crystal structure provides valuable information regarding the interactions between helical l -peptides and d -proteins, particularly elucidating a novel mode of heterochiral helix–helix interactions. Leveraging the design of d -proteins specifically targeting l -peptides or l -proteins opens up avenues for systematic exploration of the mirror-image protein universe, paving the way for a diverse range of applications.