A Large-Scale Conformational Change Couples Membrane Recruitment to Cargo Binding in the AP2 Clathrin Adaptor Complex

The AP2 adaptor complex (alpha, beta 2, sigma 2, and mu 2 sub-units) crosslinks the endocytic clathrin scaffold to PtdIns4,5P(2)-containing membranes and transmembrane protein cargo. In the locked cytosolic form, AP2's binding sites for the two endocytic motifs, Yxx Phi on the C-terminal domain of mu 2 (C-mu 2) and [ED]xxxL[LI] on sigma 2, are blocked by parts of beta 2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-mu 2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured mu 2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P(2)- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P(2)/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P(2)-containing membranes.