Structural basis for the recognition of tyrosine-based sorting signals by the μ3A subunit of the AP-3 adaptor complex

Abstract

Tyrosine-based signals fitting the YXXØ motif mediate sorting of transmembrane proteins to endosomes, lysosomes, the basolateral plasma membrane of polarized epithelial cells, and the somatodendritic domain of neurons through interactions with the homologous μ1, μ2, μ3, and μ4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXXØ signals on μ2 and μ4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXXØ signals by other members of the μ family, we solved the crystal structure at 1.85 Å resolution of the C-terminal domain of the μ3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXXØ signal (SDYQRL) from the trans-Golgi network protein TGN38. The μ3A C-terminal domain consists of an immunoglobulin-like β-sandwich organized into two subdomains, A and B. The YXXØ signal binds in an extended conformation to a site on μ3A subdomain A, at a location similar to the YXXØ-binding site on μ2 but not μ4. The binding sites on μ3A and μ2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXXØ signals. Biochemical analyses confirm the identification of the μ3A site and show that this protein binds YXXØ signals with 14-19 μm affinity. The surface electrostatic potential of μ3A is less basic than that of μ2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.

FIGURE 1.

Crystal structure of the μ3A C-terminal domain in complex with a YXXØ-encoding peptide from TGN38. A, ribbon representation of rat μ3A C-terminal domain with subdomain A in brick red, subdomain B in salmon, and the TGN38 peptide (DYQRL; stick model) in yellow. The inset shows the location of the peptide side chains on the binding site. The position of the N (N) and C (C) termini are indicated. B, stick representation of the bound peptide DYQRL (shown with carbon atoms colored yellow) superimposed on a 2F_o − F_c omit electron density map contoured at 1.5σ, and μ3A binding site amino acid residues are highlighted in stick representation (shown with carbon atoms colored gray).

FIGURE 2.

Comparison of the crystal structure of μ subunits. A, superposition of rat μ3A (red) and mouse μ1A (green; PDB code 4EN2) (22). B, superposition of μ3A (red) and rat μ2 (blue; PDB code 1BXX) (20); and C, superposition of μ3A (red) and human μ4 (orange; PDB code 3L81 (21) shown in ribbon representation. The bound peptides SYSQAAGSDSAQ on μ1A (shown with carbon atoms colored blue; oxygen colored red; nitrogen colored blue), DYQRLN on μ2 (carbon atoms colored magenta), DYQRL on μ3A (carbon atoms colored yellow), and TYKFFEQ on μ4 (carbon atoms colored green) are shown in stick representation.

FIGURE 3.

Sequence alignment of the C-terminal domains of the μ subunits. Disordered loops are in yellow letters. Arrows and cylinders represent β-strands and α-helices, respectively.

FIGURE 4.

Comparison of the binding site for the TGN38 YXXØ motif in μ2 and μ3A. A and C, surface complementarity between TGN38 peptides and μ2 (A) and μ3A (C). Surface colors for residues in contact with the TGN38 peptide are gray for hydrophobic interactions, except for Leu-175 in μ2 and Phe-181 in μ3A that are colored black. Residues forming hydrogen bonds are colored orange, except for Asp-176 in μ2 and Asp-182 in μ3A, which are colored green. The bound peptides DYQRLN on μ2 (shown with carbon atoms colored magenta; oxygen is colored red; nitrogen is colored blue; PDB code 1BXX) and DYQRL on μ3A (carbon atoms colored yellow) are shown in stick representation. B and D, two-dimensional, schematic representation of the interactions shown in A and C using LIGPLOT (48).

FIGURE 5.

Y2H analysis of the interaction of μ3A with cytosolic tails containing a YXXØ motif. A–C, yeast were co-transformed with plasmids encoding Gal4bd fused to the wild-type or Tyr-to-Ala mutant of the cytosolic tails of TGN38, CD63, or Lamp-1 constructs indicated on the left, and Gal4ad fused to wild-type or mutant μ3A constructs indicated on top of each panel. B, Y2H analysis of μ3A with mutations on the YXXØ-binding site. C, Y2H analysis of μ3A with mutations on a putative YX(FYL)(FL)E-binding site. Mouse p53 fused to Gal4bd and SV40 large T antigen (T Ag) fused to Gal4ad were used as controls. Co-transformed cells were spotted onto His-deficient (−His) or His-containing (+His) plates and incubated at 30 °C. Growth is indicative of interactions.

FIGURE 6.

ITC analysis of the interaction of μ3A with peptides containing a YXXØ motif. A, ITC of the TGN38 SDYQRL peptide (black line and solid squares) or SDAQRL peptide (gray line and gray squares) with μ3A, and of SDYQRL peptide with μ3A D182A (dashed line and open squares). B, ITC of the CD63 SGYEVM peptide (black line and solid squares) or SGAEVM peptide (gray line and gray squares) with μ3A, and of SGYEVM peptide with μ3A D182A (dashed line and open squares). The stoichiometry (N) and K_d for the μ3A-SDYQRL and for the μ3A-SGYEVM interactions are expressed as the mean ± S.E. (n = 3).

FIGURE 7.

Comparison of the surface electrostatic potential of μ subunits. A and E, two views of AP-2 complex core in the open conformation on the membrane and colored by electrostatic potential (34), with inositol 6-phosphate (IP6) in stick representation bound to a μ2 site (patch 1; E). B–D, surface representation of the orthogonal view of the structure shown in A colored by subunit (α, red; β2, blue; N-terminal domain of μ2, pale blue; σ2, green) and ribbon representation of the C-terminal domain of the indicated μ subunits superposed on the site equivalent to that of μ2 C-terminal domain. F–H, electrostatic potential of the C-terminal domain of the indicated μ subunits superposed as in B–D. Peptides with a YXXØ or a related motif are shown in stick representation colored yellow. Blue and red correspond to positive and negative potentials, respectively, with saturating color at ±5 kT/e. Black circles indicate positive patches on AP-2 for interaction with phospholipids (E) and equivalent patches on other μ subunits (F–H). I and J, comparison of the binding site for inositol 6-phosphate on the surface of μ2 (I), and the respective surface of μ3A (J), colored by electrostatic potential, showing inositol 6-phosphate and side chains in stick representation (carbon atoms in IP6 colored yellow; carbon atoms in side chains colored gray; oxygen colored red; nitrogen colored blue; phosphorus colored orange).