Structural Basis for the Recognition of Tyrosine-based Sorting Signals by the μ3A Subunit of the AP-3 Adaptor Complex

Tyrosine-based signals fitting the YXX empty set 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 mu 1, mu 2, mu 3, and mu 4 subunits of the corresponding AP-1, AP-2, AP-3, and AP-4 complexes. Previous x-ray crystallographic analyses identified distinct binding sites for YXX empty set signals on mu 2 and mu 4, which were located on opposite faces of the proteins. To elucidate the mode of recognition of YXX empty set signals by other members of the mu family, we solved the crystal structure at 1.85 angstrom resolution of the C-terminal domain of the mu 3 subunit of AP-3 (isoform A) in complex with a peptide encoding a YXX empty set signal (SDYQRL) from the trans-Golgi network protein TGN38. The mu 3A C-terminal domain consists of an immunoglobulin-like beta-sandwich organized into two sub-domains, A and B. The YXX empty set signal binds in an extended conformation to a site on mu 3A subdomain A, at a location similar to the YXX empty set -binding site on mu 2 but not mu 4. The binding sites on mu 3A and mu 2 exhibit similarities and differences that account for the ability of both proteins to bind distinct sets of YXX empty set signals. Biochemical analyses confirm the identification of the mu 3A site and show that this protein binds YXX empty set signals with 14-19 mu M affinity. The surface electrostatic potential of mu 3A is less basic than that of mu 2, in part explaining the association of AP-3 with intracellular membranes having less acidic phosphoinositides.