Molecular determinants of TRAF6 binding specificity suggest that native interaction partners are not optimized for affinity

TRAF6 is an adaptor protein involved in signaling pathways that are essential for development and the immune system. It participates in many protein–protein interactions, some of which are mediated by the C‐terminal MATH domain, which binds to short peptide segments containing the motif PxExx[FYWHDE], where x is any amino acid. Blocking MATH domain interactions is associated with favorable effects in various disease models. To better define TRAF6 MATH domain binding preferences, we screened a combinatorial library using bacterial cell‐surface peptide display. We identified 236 of the best TRAF6‐interacting peptides and a set of 1,200 peptides that match the sequence PxE but do not bind TRAF6 MATH. The peptides that were most enriched in the screen bound TRAF6 tighter than previously measured native peptides. To better understand the structural basis for TRAF6 interaction preferences, we built all‐atom structural models of the MATH domain in complex with high‐affinity binders and nonbinders identified in the screen. We identified favorable interactions for motif features in binders as well as negative design elements distributed across the motif that can disfavor or preclude binding. Searching the human proteome revealed that the most biologically relevant TRAF6 motif matches occupy a different sequence space from the best hits discovered in combinatorial library screening, suggesting that native interactions are not optimized for affinity. Our experimentally determined binding preferences and structural models support the design of peptide‐based interaction inhibitors with higher affinities than endogenous TRAF6 ligands.