Reprogramming the Specificity of a Protein Interface by Computational and Data-Driven Design

The formation of specific protein complexes in a cell is a non-trivial problem given the co-existence of thousands of different polypeptide chains. A particularly difficult case are two glutamine amidotransferase complexes (anthranilate synthase [AS] and aminodeoxychorismate synthase [ADCS]), which are composed of homologous pairs of synthase and glutaminase subunits. We have attempted to identify discriminating interface residues of the glutaminase subunit TrpG from AS, which are responsible for its specific interaction with the synthase subunit TrpEx and prevent binding to the closely related synthase subunit PabB from ADCS. For this purpose, TrpG-specific interface residues were grafted into the glutaminase subunit PabA from ADCS by two different approaches, namely a computational and a data-driven one. Both approaches resulted in PabA variants that bound TrpEx with higher affinity than PabB. Hence, we have accomplished a reprogramming of protein-protein interaction specificity that provides insights into the evolutionary adaptation of protein interfaces. [Display omitted] •The specificity of a glutaminase subunit for a synthase subunit has been inverted•The specificity switch was achieved by both a computational and a data-driven approach•The designed complexes were stable and catalytically active An important goal of protein design is the generation of non-physiological complexes between different polypeptide subunits. Hertle et al. used computational and data-driven protein design approaches to enable the binding of the glutaminase subunit from a glutamine amidotransferase (GATase) to a non-cognate synthase subunit from another GATase.