Structural basis for the recognition of two consecutive mutually interacting DPF motifs by the SGIP1 μ homology domain

FCHo1, FCHo2, and SGIP1 are key regulators of clathrin-mediated endocytosis. Their mu homology domains (mu HDs) interact with the C-terminal region of an endocytic scaffold protein, Eps15, containing fifteen Asp-Pro-Phe (DPF) motifs. Here, we show that the high-affinity mu HD-binding site in Eps15 is a region encompassing six consecutive DPF motifs, while the minimal mu HD-binding unit is two consecutive DPF motifs. We present the crystal structures of the SGIP1 mu HD in complex with peptides containing two DPF motifs. The peptides bind to a novel ligand-binding site of the mu HD, which is distinct from those of other distantly related mu HD-containing proteins. The two DPF motifs, which adopt three-dimensional structures stabilized by sequence-specific intramotif and intermotif interactions, are extensively recognized by the mu HD and are both required for binding. Thus, consecutive and singly scattered DPF motifs play distinct roles in mu HD binding.