The structure and function of the beta 2-adaptin appendage domain

Abstract

The heterotetrameric AP2 adaptor (alpha, beta 2, mu 2 and sigma 2 subunits) plays a central role in clathrin-mediated endocytosis. We present the protein recruitment function and 1.7 A resolution structure of its beta 2-appendage domain to complement those previously determined for the mu 2 subunit and alpha appendage. Using structure-directed mutagenesis, we demonstrate the ability of the beta 2 appendage alone to bind directly to clathrin and the accessory proteins AP180, epsin and eps15 at the same site. Clathrin polymerization is promoted by binding of clathrin simultaneously to the beta 2-appendage site and to a second site on the adjacent beta 2 hinge. This results in the displacement of the other ligands from the beta 2 appendage. Thus clathrin binding to an AP2-accessory protein complex would cause the controlled release of accessory proteins at sites of vesicle formation.

Fig. 1. Structural comparisons of β2- and α-appendage domains. (A) Comparison of the two C_α backbone traces of β2- (blue and red) and α-appendage (green and gold) domains. Ribbon diagrams, aligned on the N-terminal domains, coloured from N- (blue or green) to C- (red or yellow) termini. This figure, and Figures 2B and 3 were prepared using Aesop (M.E.M.Noble, unpublished). (B) Topological diagrams comparing the β2- and α-appendage domains, coloured using the same colour scheme as above.

Fig. 2. Sequence conservation between the β2- and α-appendage domains. (A) Structurally homologous residues, defined as those whose C_αs are within 3 Å in the two structures, are boxed in grey. The alignment was performed using each subdomain separately to allow for the relative rotation of C-terminal subdomains. Identities between the two sequences are coloured dark purple and conservation is indicated by pale purple. (B) Two views of the surface representation of the β2-appendage domain coloured for conservation between β2 and α in the same colour scheme as (A). The loop containing Leu747 from an adjacent molecule and the oxidized DTT molecule are shown as in Figure 3.

Fig. 3. ‘Top’ views of the C-terminal subdomains of β2- and α-appendages. The same views are used in each case for β2- (left) and α- (right) appendage domains. (A) Surfaces coloured such that the sites of favourable hydrophobic interaction are coloured dark green, sites of moderate hydrophobic interaction are coloured pale green, and sites of neutral or disfavoured hydrophobic interaction are coloured grey. The outstanding features are the hydrophobic pockets centred on the homologous tryptophan residues, occupied in β2 by Leu747 from the neighbouring molecule in the crystal and an oxidized DTT molecule. Inset: electron density for oxidized DTT as an example of overall electron density. (B) Surfaces coloured for electrostatic potential [positive (+10 kT e^–1), blue, to negative (–10 kT e^–1), red]. Surface created with GRASP (Nicholls et al., 1991). (C) Molecular detail of the surface-exposed residues on the ‘top’ of the C-terminal subdomains. (D) Effects of point mutations on protein ligand binding. Space-filling representations with colours according to the effect on protein ligand binding: red, large effect; yellow, moderate effect.

Fig. 3. ‘Top’ views of the C-terminal subdomains of β2- and α-appendages. The same views are used in each case for β2- (left) and α- (right) appendage domains. (A) Surfaces coloured such that the sites of favourable hydrophobic interaction are coloured dark green, sites of moderate hydrophobic interaction are coloured pale green, and sites of neutral or disfavoured hydrophobic interaction are coloured grey. The outstanding features are the hydrophobic pockets centred on the homologous tryptophan residues, occupied in β2 by Leu747 from the neighbouring molecule in the crystal and an oxidized DTT molecule. Inset: electron density for oxidized DTT as an example of overall electron density. (B) Surfaces coloured for electrostatic potential [positive (+10 kT e^–1), blue, to negative (–10 kT e^–1), red]. Surface created with GRASP (Nicholls et al., 1991). (C) Molecular detail of the surface-exposed residues on the ‘top’ of the C-terminal subdomains. (D) Effects of point mutations on protein ligand binding. Space-filling representations with colours according to the effect on protein ligand binding: red, large effect; yellow, moderate effect.

Fig. 4. Binding partners of β2 appendage and β2 appendage+hinge. Proteins bound to GSTβ2 appendage, β2 appendage+hinge, α appendage and Amph2SH3 [control in (B)] were analysed by Coomassie Blue staining (A) and immunoblotting with antibodies (B) against clathrin, AP180, epsin, eps15, amphiphysin1 and synapsin (an abundant protein in brain that has been proposed to bind to the SH3 domains of the amphiphysins) as a control to show that ligand binding to the appendages is specific. (C) Effects of transfection of constructs expressing the α appendage, β2 appendage, β2 appendage+hinge, and β2 appendage+hinge containing the mutation Y888V domains on transferrin endocytosis in COS7 fibroblasts. Expression levels of each construct were equal as judged by the intensity of immunofluorescence.

Fig. 5. The protein–protein interaction site on the β2-appendage domain. (A) GSTβ2-appendage domain point mutants, GSTα appendage and GSTAmph2 SH3 (negative control) were tested for their ability to bind proteins in ‘pull down’ experiments from brain cytosol detected by immunoblotting. (B) Overlay assays. Lanes 1 and 2 show overlays of rat brain cytosolic proteins with the α- and β2-appendage domains. Appendage domains were detected with Ra5.2 (α) or Ra2.2 (β). Direct detection of proteins in rat brain cytosol with specific antibodies are shown in the remaining lanes.

Fig. 6. GSTβ2 appendage+hinge but not GSTβ2 appendage can drive clathrin cage formation. Clathrin at a final concentration of 0.7 µM was incubated on its own (lane 1) or in the presence of a 4-fold (lane 2) or 1.5-fold (lane 3) molar excess of GSTβ2 appendage+hinge, or an 8-fold (lane 4) or 4-fold (lane 5) molar excess of GSTβ2 appendage. Lanes 6 and 7 contained the same amounts of the GST proteins as in lanes 2 and 4, respectively, but no clathrin. Samples were centrifuged at 100 000 g and pelletable material assayed by SDS–PAGE followed by Coomassie Blue staining (A) or by negative stain electron microscopy (B and C). GSTβ2 appendage+hinge but not GSTβ2 appendage can drive clathrin cage formation, as can be seen by the increased amount of pelletable clathrin in lanes 2 and 3 compared with lanes 1, 4 and 5 (A). The electron micrographs in (B) and (C) show the pelletable material to be cages and not non-specific aggregates.