Abstract
Protein-protein interaction specificity is often encoded at the primary sequence level. However, the contributions of individual residues to specificity are usually poorly understood and often obscured by mutational robustness, sequence degeneracy, and epistasis. Using bacterial toxin-antitoxin systems as a model, we screened a combinatorially complete library of antitoxin variants at three key positions against two toxins. This library enabled us to measure the effect of individual substitutions on specificity in hundreds of genetic backgrounds. These distributions allow inferences about the general nature of interface residues in promoting specificity. We find that positive and negative contributions to specificity are neither inherently coupled nor mutually exclusive. Further, a wild-type antitoxin appears optimized for specificity as no substitutions improve discrimination between cognate and non-cognate partners. By comparing crystal structures of paralogous complexes, we provide a rationale for our observations. Collectively, this work provides a generalizable approach to understanding the logic of molecular recognition.
Highlights
Protein-protein interactions underlie most cellular processes and are the basis of established and emerging therapies such as monoclonal antibodies (Weiner et al, 2010), chimeric antigen receptor T cells (Brentjens et al, 2013), and stapled peptide drugs (Walensky and Bird, 2014)
To examine insulation between paralogous toxin-antitoxin loci, we focused on two closely-related systems from the widespread ParD-ParE family
We previously showed that ParD antitoxins are highly specific for their cognate ParE toxins, and that interaction specificity is controlled by a discrete set of coevolving residues that map to the protein-protein interface formed by ParD and ParE (Aakre et al, 2015)
Summary
Protein-protein interactions underlie most cellular processes and are the basis of established and emerging therapies such as monoclonal antibodies (Weiner et al, 2010), chimeric antigen receptor T cells (Brentjens et al, 2013), and stapled peptide drugs (Walensky and Bird, 2014). To prevent non-specific and potentially detrimental interactions, proteins must discriminate between cognate and non-cognate interaction partners. This is often achieved via molecular recognition or non-covalent interactions between protein surfaces. The structural space of interfaces is degenerate, with nearly 90% of native interfaces having a close structural neighbor with a highly similar backbone geometry (Gao and Skolnick, 2010). This problem is acute for cells that encode paralogous protein families, whose members can share significant structural and sequence similarity. Prior work has shown that the specificity of many paralogous protein families is determined by a subset of residues that typically map to the interface formed by a given protein and its binding partner(s) (Aakre et al, 2015; Ovchinnikov et al, 2014; Skerker et al, 2008)
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