RESUMEN
Short sequences that mediate interactions with modular binding domains are ubiquitous throughout eukaryotic proteomes. Networks of Short Linear Motifs (SLiMs) and their corresponding binding domains orchestrate many cellular processes, and the low mutational barrier to evolving novel interactions provides a way for biological systems to rapidly sample selectable phenotypes. Mapping SLiM binding specificity and the rules that govern SLiM evolution is fundamental to uncovering the pathways regulated by these networks and developing the tools to manipulate them. We used high-throughput screening of the human proteome to identify sequences that bind to the Enabled/VASP homology 1 (EVH1) domain of the postsynaptic density scaffolding protein Homer1. In doing so, we expanded current understanding of the determinants of Homer EVH1 binding preferences and defined a new motif that can facilitate the discovery of additional Homer-mediated interactions. Interestingly, the Homer1 EVH1 domain preferentially binds to sequences containing an N-terminally overlapping motif that is bound by the paralogous family of Ena/VASP actin polymerases, and many of these sequences can bind to EVH1 domains from both protein families. We provide evidence from orthologous EVH1 domains in pre-metazoan organisms that the overlap in human Ena/VASP and Homer binding preferences corresponds to an incomplete divergence from a common Ena/VASP ancestor. Given this overlap in binding profiles, promiscuous sequences that can be recognized by both families either achieve specificity through extrinsic regulatory strategies or may provide functional benefits via multi-specificity. This may explain why these paralogs incompletely diverged despite the accessibility of further diverged isoforms.
RESUMEN
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.