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

Abstract

SummaryThe AP2 adaptor complex (α, β2, σ2, and μ2 subunits) crosslinks the endocytic clathrin scaffold to PtdIns4,5P2-containing membranes and transmembrane protein cargo. In the “locked” cytosolic form, AP2's binding sites for the two endocytic motifs, YxxΦ on the C-terminal domain of μ2 (C-μ2) and [ED]xxxL[LI] on σ2, are blocked by parts of β2. Using protein crystallography, we show that AP2 undergoes a large conformational change in which C-μ2 relocates to an orthogonal face of the complex, simultaneously unblocking both cargo-binding sites; the previously unstructured μ2 linker becomes helical and binds back onto the complex. This structural rearrangement results in AP2's four PtdIns4,5P2- and two endocytic motif-binding sites becoming coplanar, facilitating their simultaneous interaction with PtdIns4,5P2/cargo-containing membranes. Using a range of biophysical techniques, we show that the endocytic cargo binding of AP2 is driven by its interaction with PtdIns4,5P2-containing membranes.

Molecular Structure, Function, and Dynamics of Clathrin-Mediated Membrane Traffic

“…One of two splice forms of synaptojanin 1 also has clathrin and AP2-binding motifs, distal to the proline-rich segment. (Yu et al 2010), superposed onto the structure of the open AP2 core (PDB 2XA7) (Jackson et al 2010). The tyrosine motif is represented as sticks; the DEP domain (dark gray) and m2-C (lighter gray) as ribbons; the rest of the AP2 core as a surface.…”

“…Indeed, it would be substantially less likely that a diffusing transmembrane protein could enter a constricted, nearly mature pit than a gently domed one or a planar structure. Clathrin binding to AP2 is linked to its transition from a locked to an open conformation, and thus can couple cargo capture and lattice assembly (Rapoport et al 1997;Jackson et al 2010). …”

“…The heterotetramers of AP2 and AP1 have at least two distinct states-a closed or "locked" state and one or more open states (Rapoport et al 1997;Collins et al 2002;Jackson et al 2010). In the closed state, two well-studied cargo-binding sites are buried (Fig.…”

Identification and Dynamics of Arabidopsis Adaptor Protein-2 Complex and Its Involvement in Floral Organ Development

“…The null mutant of the m-subunit still accumulates the a-and s-subunits, and the double mutant lacking m-and a-subunits is embryo lethal (Gu et al, 2013). It has been suggested that the a/s-and b/m-subunits have the potential to form large/small subunit heterodimers (Collins et al, 2002;Jackson et al, 2010), which may partially compensate for the loss of AP-2 (Gu et al, 2013). Such mechanisms might complement the lack of AP-2 function in the ap2m mutant plants.…”

Endocytic Accessory Factors and Regulation of Clathrin-Mediated Endocytosis

“…The distribution of PIP2 in the inner leaflet of the mammalian plasma membrane is uniform in nonexcitable cells (Antonescu et al 2010) and AP2 has a relatively low affinity for PIP2, leading to the suggestion that AP2 "samples" the plasma membrane (Cocucci et al 2012). On binding PIP2 the AP2 complex undergoes a dramatic structural rearrangement and unfurls to reveal cargo and clathrin-binding motifs (Jackson et al 2010). If the AP2 complex then successfully binds receptor cargo and clathrin further PIP2-binding motifs are revealed, association with the membrane strengthens and nucleation of a nascent CCP begins (Jackson et al 2010).…”