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...

Dysregulation of the calcitonin gene-related peptide (CGRP), a potent vasodilator, is directly implicated in the pathogenesis of migraine. CGRP binds to and signals through the CGRP receptor (CGRP-R), a heterodimer containing the calcitonin receptor-like receptor (CLR), a class B GPCR, and RAMP1, a receptor activity-modifying protein. We have solved the crystal structure of the CLR/RAMP1 N-terminal ectodomain heterodimer, revealing how RAMPs bind to and potentially modulate the activities of the CLR GPCR subfamily. We also report the structures of CLR/RAMP1 in complex with the clinical receptor antagonists olcegepant (BIBN4096BS) and telcagepant (MK0974). Both drugs act by blocking access to the peptide-binding cleft at the interface of CLR and RAMP1. These structures illustrate, for the first time, how small molecules bind to and modulate the activity of a class B GPCR, and highlight the challenges of designing potent receptor antagonists for the treatment of migraine and other class B GPCR-related diseases.

GSK3beta was identified as the kinase that phosphorylates glycogen synthase but is now known to be involved in multiple signaling pathways. GSK3beta prefers prior phosphorylation of its substrates. We present the structure of unphosphorylated GSK3beta at 2.7 A. The orientation of the two domains and positioning of the activation loop of GSK3beta are similar to those observed in activated kinases. A phosphate ion held by Arg 96, Arg 180 and Lys 205 occupies the same position as the phosphate group of the phosphothreonine in activated p38gamma, CDK2 or ERK2. A loop from a neighboring molecule in the crystal occupies a portion of the substrate binding groove. The structure explains the unique primed phosphorylation mechanism of GSK3beta and how GSK3beta relies on a phosphoserine in the substrate for the alignment of the beta- and alpha-helical domains.