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

Abstract

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. computational design | recognition specificity | promiscuity | protein interaction domains | interface evolution M any protein-protein interactions are mediated by small modular protein recognition domains (1). These interaction modules, such as PDZ, SH3, and WW domains, generally recognize their protein partners using a structurally conserved binding site, and there are often tens or even hundreds of proteins containing a given type of recognition domain expressed simultaneously in a cell or organism (2). This repeated use of recognition modules with conserved structures poses the following question: how do cells maintain the specificity of binding interactions when so many members of the same domain family are present? Moreover, to what extent do different domain family members in fact have distinct or overlapping preferences for binding their partners? Addressing these questions is of considerable importance, because a significant fraction of protein interactions in a cell is mediated a limited number of protein interaction domain families (1). Despite a large amount of information on the biochemical recognition preferences of many domain members in vitro (3-5), much less is known about the actual extent of specificity an...

Epistasis in protein evolution

“…Second, epistasis can yield evolutionary “dead‐ends” in sequence space, from which a potentially beneficial mutation is not immediately accessible; in such cases, a relaxation of selection or even selection for other protein properties is necessary before a trajectory is opened to a superior optimum . Third, epistasis can cause a mutation that confers or improves a function in one protein to have no effect or even be strongly deleterious in a related protein; as a result, attempts to leverage natural sequence variation or experimental observations to predict mutational effects or engineer proteins with desired properties often fail . These issues highlight why characterizing epistasis—including the breadth of its effect, its mechanistic underpinnings, and its evolutionary impact—is important for our basic understanding of protein biochemistry and evolution.…”

Using Evolution to Guide Protein Engineering: The Devil IS in the Details

“…Epistasis has confounded many attempts to design proteins. For example, epistasis was observed during efforts to transplant protein-protein interfaces among PDZ domains (68). Epistasis is now recognized to be a dominant feature in the evolution of natural proteins such the influenza A surface proteins (69) and TEM-1 b-lactamase (70).…”

Distinct Roles for Conformational Dynamics in Protein-Ligand Interactions

“…Thus, PDZ/peptide interactions feature epistasis (i.e. context dependence) and therefore specificity results from a precise combination of interactions throughout the entire binding interface (Ernst et al, 2010; Ernst et al, 2009; Liu et al, 2013; Melero et al, 2014; Shepherd et al, 2011; Stiffler et al, 2007). Structural analyses of PDZ complexes provide ample support for this idea (Ernst et al, 2014; Lee and Zheng, 2010).…”