Nonnatural protein–protein interaction-pair design by key residues grafting

Protein–protein interface design is one of the most exciting fields in protein science; however, designing nonnatural protein–protein interaction pairs remains difficult. In this article we report a de novo design of a nonnatural protein–protein interaction pair by scanning the Protein Data Bank for suitable scaffold proteins that can be used for grafting key interaction residues and can form stable complexes with the target protein after additional mutations. Using our design algorithm, an unrelated protein, rat PLCδ1-PH (pleckstrin homology domain of phospholipase C-δ1), was successfully designed to bind the human erythropoietin receptor (EPOR) after grafting the key interaction residues of human erythropoietin binding to EPOR. The designed mutants of rat PLCδ1-PH were expressed and purified to test their binding affinities with EPOR. A designed triple mutation of PLCδ1-PH (ERPH1) was found to bind EPOR with high affinity (KD of 24 nM and an IC50 of 5.7 μM) both in vitro and in a cell-based assay, respectively, although the WT PLCδ1-PH did not show any detectable binding under the assay conditions. The in vitro binding affinities of the PLCδ1-PH mutants correlate qualitatively to the computational binding affinities, validating the design and the protein–protein interaction model. The successful practice of finding a proper protein scaffold and making it bind with EPOR demonstrates a prospective application in protein engineering targeting protein–protein interfaces.