Abstract

Membrane proteins are packaged for transport between the different membrane compartments of eukaryotic cells into small vesicles formed by an elaborate system of cytoplasmic proteins. Selection of cargo for vesicle formation at the plasma membrane (endocytosis) is generally mediated directly or indirectly by the heterotetrameric clathrin adaptor complex AP2 , which binds short sequence recognition motifs of two types, YxxΦ (tyrosine-based motif, where Φ is a hydrophobic residue) and [DE]xxxLL (acidic dileucine motif). The structure of the 200kDa AP2 “core” crystallised in the absence of peptides showed a closed conformation, with binding sites for both types of motifs blocked, and indeed AP2 in solution does not bind motif peptides. AP2 is activated by binding to negatively charged membranes containing phosphatidylinositol-(4,5)-bisphosphate. We were able to trap the activated “open” conformation in crystals grown with a YxxΦ peptide, and this structure shows a large conformational change compared to the closed “locked” conformation, with the YxxΦ-binding domain moving out of the “bowl” formed by the other subunits. This places both peptide sites on the positively-charged face of the complex, allowing simultaneous interaction with cargo motifs and the membrane. Thus AP2 functions as a plasma membrane-activated switch for endocytic cargo recognition.