Quantification of the transferability of a designed protein specificity switch reveals extensive epistasis in molecular recognition

Significance Specific interactions between proteins control the function of essentially all cellular processes. Despite the importance of interaction specificity, it is unclear how structurally similar proteins achieve their unique recognition preferences. Here, we redesign the specificity of a protein binding domain and quantify the extent to which the designed specificity switch can be transferred to homologous domains. We show that identical mutations in structurally similar domains have a wide range of effects on specificity. We apply a structure-based computational model that recapitulates this context dependence. Our findings show how subtle structural differences between homologous domains contribute to their unique specificities. The differential responses to similar mutation observed here could help explain how families of recognition domains have evolved diverse new interactions. Reengineering protein–protein recognition is an important route to dissecting and controlling complex interaction networks. Experimental approaches have used the strategy of “second-site suppressors,” where a functional interaction is inferred between two proteins if a mutation in one protein can be compensated by a mutation in the second. Mimicking this strategy, computational design has been applied successfully to change protein recognition specificity by predicting such sets of compensatory mutations in protein–protein interfaces. To extend this approach, it would be advantageous to be able to “transplant” existing engineered and experimentally validated specificity changes to other homologous protein–protein complexes. Here, we test this strategy by designing a pair of mutations that modulates peptide recognition specificity in the Syntrophin PDZ domain, confirming the designed interaction biochemically and structurally, and then transplanting the mutations into the context of five related PDZ domain–peptide complexes. We find a wide range of energetic effects of identical mutations in structurally similar positions, revealing a dramatic context dependence (epistasis) of designed mutations in homologous protein–protein interactions. To better understand the structural basis of this context dependence, we apply a structure-based computational model that recapitulates these energetic effects and we use this model to make and validate forward predictions. Although the context dependence of these mutations is captured by computational predictions, our results both highlight the considerable difficulties in designing protein–protein interactions and provide challenging benchmark cases for the development of improved protein modeling and design methods that accurately account for the context.