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.

TRAF6 is an adapter protein and E3 ubiquitin ligase that is involved in signaling downstream of cell receptors important for development and immune system activation and maintenance. TRAF6 participates in hundreds of protein-protein interactions, some of which are mediated by a C-terminal MATH domain that recruits TRAF6 to cell-surface receptors and associated proteins. The TRAF6 MATH domain binds to short peptide segments containing the motif PxExx[FYWHDE], where x is any amino acid. Blocking TRAF6 interactions is associated with favorable effects in a variety of disease models. To better define TRAF6 binding preferences, we generated a bacterial cell-surface peptide display library to sample the TRAF6 motif sequence space. We performed sorting experiments to identify 236 of the best TRAF6-interacting peptides and a set of 1,200 peptides that match the sequence PxE but do not bind TRAF6. Selected binders tested by single-clone bacterial display titrations and by bio-layer interferometry bound TRAF6 tighter than previously measured native peptides. We built all-atom structural models of the TRAF6 MATH domain in complex with high-affinity binders and motif-matching nonbinders identified by screening to elucidate the structural basis for TRAF6 interaction preferences. We identified motif features that favor binding to TRAF6 and also negative design elements, distributed across the motif, that can disfavor or preclude binding. Searching the human proteome for matches to the library screening-defined binding motif revealed that most known, biologically relevant TRAF6 binders occupy a different sequence space from the most enriched hits that we discovered in combinatorial library screening. Our experimentally determined binding preferences and structural models can support the design of peptide-based interaction inhibitors with higher affinities than endogenous TRAF6 ligands.