A structural explanation for the binding of endocytic dileucine motifs by the AP2 complex

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Abstract
The movement of most transmembrane proteins between the various membranes of the cell is directed by short linear amino acid sequences in their cytoplasmic portions. These sequence motifs are bound by protein components of transport vesicle coats thereby incorporating the transmembrane proteins as vesicle cargo. In the case of clathrin-coated vesicles (CCVs) the motifs are recognised by the AP complexes or GGA clathrin adaptors. The clathrin adaptor AP2 recognizes two major classes of endocytic motifs including an acidic dileucine motif. In this study, Kelly et al. present the crystal structure of AP2 in a complex with the dileucine motif of a cargo protein thereby revealing the mechanism of cargo-adaptor recognition. During clathrin-mediated endocytosis, cargo proteins are recognized by clathrin adaptors. The clathrin adaptor AP2 recognizes two major classes of endocytic motifs, including an acidic dileucine motif. This study presents the crystal structure of AP2 in complex with the diceucine motif of a cargo protein, thereby revealing the mechanism of cargo–adaptor recognition. Most transmembrane proteins are selected as transport-vesicle cargo through the recognition of short, linear amino-acid motifs in their cytoplasmic portions by vesicle coat proteins. For clathrin-coated vesicles, the motifs are recognized by clathrin adaptors. The AP2 adaptor complex (subunits α, β2, μ2 and σ2) recognizes both major endocytic motifs: YxxΦ motifs1 (where Φ can be F, I, L, M or V) and [ED]xxxL[LI] acidic dileucine motifs. Here we describe the binding of AP2 to the endocytic dileucine motif from CD4 (ref. 2). The major recognition events are the two leucine residues binding in hydrophobic pockets on σ2. The hydrophilic residue four residues upstream from the first leucine sits on a positively charged patch made from residues on the σ2 and α subunits. Mutations in key residues inhibit the binding of AP2 to ‘acidic dileucine’ motifs displayed in liposomes containing phosphatidylinositol-4,5-bisphosphate, but do not affect binding to YxxΦ motifs through μ2. In the ‘inactive’ AP2 core structure3 both motif-binding sites are blocked by different parts of the β2 subunit. To allow a dileucine motif to bind, the β2 amino terminus is displaced and becomes disordered; however, in this structure the YxxΦ-binding site on μ2 remains blocked.