Accurate de novo design of heterochiral protein-protein interactions

Abiotic D-proteins that target genetically encodable L-proteins are of considerable biotechnological interest, however, the structural principles governing such heterochiral proteinprotein interactions are mainly unknown. Here, we describe the de novo design of D-proteins that bind to a specific surface region of a target L-protein. The designer D-binder proteins target one designer peptide and two human proteins of therapeutic interest with subnanomolar affinity before affinity maturation, exhibiting high enantiomeric specificity and target specificity, and inhibit target cellular signaling in a concentration-dependent manner. The designer D-binder proteins are hyperstable and protease resistant. The crystal structure of a designer heterochiral protein complex is nearly identical to the design model, and provides new insights into the interactions between L- and D-proteins. The targeted design of protein-targeting D-proteins with atomic-level accuracy enables rational exploration of the abiotic mirror-image protein universe and paves the way for a wide variety of applications.