Accurate de novo design of heterochiral protein–protein interactions

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.