The solution structure of pGolemi, a high affinity Mena EVH1 binding miniature protein, suggests explanations for paralog-specific binding to Ena/VASP homology (EVH) 1 domains

Abstract

Ena/VASP homology 1 (EVH1) domains are polyproline binding domains that are present in a wide range of adaptor proteins, among them Ena/VASP proteins involved in actin remodeling and axonal guidance. The interaction of ActA, a transmembrane protein from the food-borne pathogen Listeria monocytogenes, with EVH1 domains has been shown to be crucial for recruitment of the host's actin skeleton and, as a consequence, for the infectivity of this bacterium. We present the structure of a synthetic high-affinity Mena EVH1 ligand, pGolemi, capable of paralog-specific binding, solved by NMR spectroscopy. This peptide shares the common pancreatic peptide fold with its scaffold, avian pancreatic peptide, but shows pivotal differences in the aminoterminus. The interplay of spatial fixation and flexibility appears to be the reason for its high affinity towards Mena EVH1. Combined with earlier investigations, our structural data shed light on the specificity determinants of pGolemi and the importance of additional binding epitopes around the residues Thr74 and Phe32 on EVH1 domains regulating paralog specificity. Our results are expected to facilitate the design of other high-affinity, paralog-specific EVH1 domain ligands, and serve as a fundament for the investigation of the molecular mode of action of EVH1 domains.

A modular toolkit to inhibit proline-rich motif–mediated protein–protein interactions

“…Further interactions of flanking residues located outside the core motif contribute substantially to both affinity and specificity. Incorporation of nonnatural amino acids in place of such specificity-determining residues is therefore often beneficial for binding (4)(5)(6)(7)(8)(9). However, peptide ligands display a number of disadvantages when used as competitors, among them metabolic instability and often low cell permeability.…”