Peptide-in-groove interactions link target proteins to the β-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 ␤-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, ␤-arrestin 2 and the ␤-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 ␤-propellers recognize specific target proteins. C haracteristic ''interaction domains,'' of various conserved designs, often are responsible for specific protein associations in sorting and signaling pathways. One example is the ␤-propeller formed by seven ''WD40 repeats''-modules, such as those in heterotrimeric G proteins, that contain about 40 residues with a tryptophan (W) and an aspartic acid (D) at defined positions (1-3). Each repeat forms a four-strand ␤-sheet-one blade of the propeller-like domain. The ''terminal domain'' of clathrin has an essentially identical propeller structure, although it lacks tryptophans and aspartic acids at the canonical positions in most of its seven blades (4).Clathrin is a three-legged (''triskelion''-shaped) molecule (5, 6) that forms cage-like enclosures (7-9) and drives vesiculation of cellular membranes (10-13). Each clathrin leg is an Ϸ1,600-aa heavy chain, with the Ϸ330-residue, amino-terminal ␤-propeller domain at its tip (14-16). The terminal domain connects to a set of about 42 ␣-zigzags, which forms the 450-Å-long extended part of the leg and which ends in a small trimerization domain at the carboxyl terminus (4, 17). The assembling clathrin lattice recruits membrane-anchored cargo to the coat through adaptor proteins, which interact with the terminal domain (18-21). One particular adaptor, ␤-arrestin, directs internalization of ␤ 2 -adrenergic receptors through the clathrin-endocytic pathway (22-24). ␤-Arrestin attaches to clathrin through a short peptide near its carboxyl terminus (25). The crystal structure of ␣-arrestin, the corresponding protein in the visual system, shows that the clathrin-interacting segment in nonvisual arrestins would be part of a carboxyl-terminal flexible loop (26). Clathrin residues that influence binding with this extended element from ␤-arrestins lie in the groove between blades 1 and 2 of the terminal domain (4, 25).The structures reported here show that the interacting segment of ␤-arrestin runs along the groove and also demonstrate that ␤-subunits of the heterotetrameric clathrin adaptors interact with the same site...

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

“…Although the overall identity of the full-length proteins is significantly lower, ␤2 (170 -518) shares 92.3% sequence identity to ␤1 (170 -518), 27.7% to ␤3A (197-569), 28.8% to ␤3B (192-574), and 28.5% to ␤4 (169 -510). Importantly, the homologous region identified here is N-terminal to the flexible hinge region containing the clathrin box signals of ␤1, ␤2, ␤3A, and ␤3B (ter Haar et al, 2000). In support of our finding, recent computational analyses on HEAT and ARM repeat structures suggested ␤-adaptins and ␤-COP to be HEAT repeat containing proteins (Traub, 1997;Andrade et al, 2001).…”

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

“…The terminal domain, protruding inward from the clathrin lattice, is free to contact adaptors and other membrane-interacting components (ter Haar et al 1998(ter Haar et al , 2000Drake et al 2000;Drake and Traub 2001;Miele et al 2004). It projects toward the axis of a vertex three lattice points removed from the one on which its triskelion is centered ( Fig.…”

“…Although the azig-zag linker connecting it to the main part of the clathrin leg is relatively stiff, the "fuzziness" of the terminal domain in the cryoEM reconstruction indicates some variability in its position with respect to the lattice as a whole, probably because of flexibility in the ankle regions that connect terminal domains to distal legs. The terminal domain interacts with heterotetra-meric adaptors through a peptide motif in the long, flexible "hinge" of the b-chain having the consensus sequence LFXFD/E ("clathrin box"; F represents a hydrophobic residue) (Shih et al 1995;ter Haar et al 2000). Clathrin-box peptides bind in a groove between blades 1 and 2 of the terminal-domain b-propeller (ter Haar et al 2000;Miele et al 2004).…”

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

“…CME inhibition is based on the ability of Pitstop compounds to interfere with the function of the clathrin TD, a seven-bladed ␤-propeller, as a recruitment hub for accessory proteins harboring clathrin box motifs (31) including AP and GGA adaptors, Eps15, epsins, amphiphysin, SNXs, or CALM (1,3,5,11,32,33). Although Pitstop-2 has been shown to inhibit CME of transferrin and HIV uptake via arrest of CCP dynamics (31), its activity with respect to intracellular membrane traffic has not been explored.…”