Peptide-in-groove interactions link target proteins to the beta-propeller of clathrin

Abstract

The "WD40" domain is a widespread recognition module for linking partner proteins in intracellular networks of signaling and sorting. The clathrin amino-terminal domain, which directs incorporation of cargo into coated pits, is a beta-propeller closely related in structure to WD40 modules. The crystallographically determined structures of complexes of the clathrin-terminal domain with peptides derived from two different cargo adaptors, beta-arrestin 2 and the beta-subunit of the AP-3 complex, reveal strikingly similar peptide-in-groove interactions. The two peptides in our structures contain related, five-residue motifs, which form the core of their contact with clathrin. A number of other proteins involved in endocytosis have similar "clathrin-box" motifs, and it therefore is likely that they all bind the terminal domain in the same way. We propose that a peptide-in-groove interaction is an important general mode by which beta-propellers recognize specific target proteins.

Figure 1

Structures of clathrin-terminal domain complexed with clathrin-box peptides from β-arrestin 2 (a and b) and β3-hinge of AP-3 (c and d). (a and c) Ribbon diagrams of the complexes. The representation is a “top” view of the td40 fragment (residues 3–359) looking from the membrane toward the surrounding clathrin coat. The propeller blades are numbered 1–7; each blade contains four antiparallel β-strands, labeled a–d. Ordered portions of the bound peptides from β-arrestin 2 and β3-hinge (of sequence DTNLIEFE and VSLLDLD) are in green. The figure was made with ribbons (31). (b and d) Surface representation of the complexes. The images of b and d have been rotated approximately 90° toward the viewer. The map shows positive (blue), negative (red), and hydrophobic/neutral patches (white) projected onto the surface representation. The figure was made with grasp (32).

Figure 2

Alignment of sequences found in proteins that bind clathrin (, , –35, 38, 39). The comparison delineates a conserved clathrin-box motif, a consensus noted in ref. . The clathrin box is surrounded in each of these proteins by unrelated residues.

Figure 3

Close-up view of the peptide-in-groove interactions between the clathrin box in the peptide of β-arrestin 2 and the clathrin-terminal domain. The residues that contribute to the interaction between the β-arrestin 2 peptide and the clathrin groove between blades 1 and 2 are labeled by single-letter code and by the positions in their respective sequences. The dashed lines show hydrogen bonds between the backbones of blade 1d and bound peptide and between peptide backbone and the side chain of clathrin Q89. The locations for the two hydrophobic (gray) and polar (rose) pockets in the clathrin groove are approximate.

Figure 4

Schematic representation of the network of contact arrays of target proteins and clathrin within a coat. Key features illustrated are: 1, the contact between the clathrin terminal domain (yellow) and the clathrin-box sequences (black) in β-arrestins (green) and the β-chain of AP complexes (blue); 2, a possible additional contact (see text) between β-arrestins and terminal domain; 3, a contact, inferred from mutational studies between the carboxyl-terminal part of β-arrestins and the β-subunit of AP-2. APs are generalized adaptors for sorting signals (YppØ, LL) in various receptors; β-arrestins are specific adaptors for seven-transmembrane, G protein-coupled receptors (dark blue). The membrane is shown in gray; the framework of the clathrin coat is shown in red.