Dissecting the Engrailed homeodomain-DNA interaction by phage-displayed shotgun scanning

Phage-displayed alanine shotgun scanning was used to dissect contributions by engrailed homedomain (En-HD) residues 17 through 46, which indirectly influence recognition of DNA. The relative contributions of such indirect contacts, quantified by shotgun scanning, highlight previously unexplored En-HD residues. Two motifs dominate En-HD function in this region. First, two surface-exposed aromatic residues (F20 and Y25) bracket the hydrophobic core. Second, two sets of turn-forming residues are highlighted, including carboxamide-requiring residues E22/N23 and a leucine/isoleucine splint. The En-HD hydrophobic core exhibits a surprising degree of malleability, as demonstrated by homolog shotgun scanning. Most selectants from in vitro shotgun scanning mirror the consensus human homeodomain sequence. Thus, natural evolution and in vitro selection use similar selection criteria: affinity, specificity, and stability. However, homolog shotgun scanning identified mutations capable of improving the affinity and specificity of En-HD.