[WS4] APPA/PSSJ Joint Workshop Toward International Cooperation in Protein Science

[WS4-04] Putting ENAH EVH1 binders in context

^○Amy E. Keating¹, Theresa Hwang², Meucci Watchman Ilunga², Robert A. Grant² (1.Professor of Biology and Biological Engineering, Massachusetts Institute of Technology, 2.Department of Biology, Massachusetts Institute of Technology)

Many protein-protein interactions involve short, linear motifs (SLiMs) binding to folded protein domains. High-resolution structures typically show just a few SLiM residues engaging with the domain, and it is unclear how such minimal interactions can encode sufficient information to direct the appropriate interaction specificity. One possibility is that the sequence context surrounding SLiMs modulates binding. We used a proteome-wide screen to test this idea for SLiMs that bind to the ENAH EVH1 domain. We identified three ways in which proximal and distal flanking regions enhance binding affinity and/or specificity. Crystal structures of the ENAH EVH1 domain bound to extended peptides that encompass SLiMs reveal how SLiM-flanking regions impact binding. The highest affinity binder from our screen exhibits an unanticipated binding mode that stabilizes a unique conformation adopted only by the EVH1 domain of ENAH and not its closely related paralogs, providing a novel specificity mechanism. Guided by our analyses of native binders, we designed the tightest known ENAH EVH1 binder to yet be reported, which will be useful for dissecting the unique functions of ENAH in regulating cell motility and other processes.