A groovy new structure.

R ter Haar et al. (1) showed that the N-terminal domain of clathrin, the major protein component of clathrin-coated vesicles, is made up of the increasingly commonly recognized b propeller fold. In this issue of PNAS, ter Haar et al. (2) now show that peptides from two clathrin adapters, b-arrestin 2 and the b subunit of AP-3, bind to a groove on the b propeller surface. This appears to be a novel method of protein– protein binding for b propeller structures that adds a new facet to the ingenious ways that these structures have devised for binding protein ligands, sugars, prosthetic groups, and ions. Clathrin-coated vesicles are one class of transport vesicles that have a cage of proteins covering the cytosolic surface. Clathrin is a protein complex that is made up of three heavy and three light chains. Together, these proteins form a three-legged structure called a triskelion. The triskelions assemble to form a network of hexagons and pentagons that make up the coated pits on the cytoplasmic surface of the membrane. The amino terminal portions of each heavy chain turn inward and form a secondary shell inside the cage-like clathrin structure. As the clathrin-coated vesicles assemble, they recruit membraneanchored proteins to the cage-like structure through adapter proteins that interact with the N-terminal, b propeller domain. As ter Haar et al. (2) show, two adapter proteins, b-arrestin and AP-3, bind in part through interaction of short peptides with a groove between the first two propeller structural repeats called blades. It is likely that two other adapter proteins, AP-1 and AP-2, also bind to the groove between blades 1 and 2 (2). It is interesting that both peptides bind to the same site on the seven-bladed clathrin propeller because earlier work suggested that b-arrestin and AP-2 would bind to different sites (3). However, the holoprotein may bind to additional regions besides those identified for these peptides. Although the proteins can, in principle, compete for the same binding sites, this is not likely to be important in vivo because there is more clathrin than either b-arrestin or the AP proteins. They are not, therefore, likely to saturate the clathrin binding sites. Some clathrin ligands can interact with each other and, the authors suggest, might cooperate in binding to the coated vesicles. b propellers turn out to be a common fold that has been highly exploited over the past three billion years of evolution. These proteins have a symmetric architecture made up of 4–8 structural repeats. Each of these repeats or propeller blades is made up of four antiparallel b strands radiating outward from a central axis. All of the proteins with this fold have a high percentage of large andyor aromatic hydrophobic residues that form the contact surface between the blades. The toroidal structure has a narrower end (often called the top) and a wider end (the bottom). In addition, there is a central tunnel that varies in shape and diameter. The mean diameter of the tunnel increases as the number of blades increases. Its degree of cylindrical symmetry is directly related to the degree of structural similarity among the blades. The b propeller proteins can be divided into several families. The most diverse and currently largest single family is characterized by the so-called WD sequence repeat. The only WD repeat protein whose structure has been determined is the b subunit of heterotrimeric G proteins (5–7). However, the highly conserved sequence of the WD repeat proteins makes it very likely that they all have a propeller structure, but with different numbers of blades. The WD repeat consists of a near constant length region defined by a pattern of amino acid residues typically bracketed by the dipeptides GH and WD (4). This sequence pattern has a one-toone correspondence to the inner three b strands of the structural repeat. The outermost strand (d strand) is encoded in a variable length region with sequences specific to the different WD repeat functional subfamilies. The most conserved feature of the WD repeat proteins is an aspartic acid residue six positions N-terminal to the WD that is present in 86% of the WD repeats (8). In the b subunit of the heterotrimeric G proteins, it forms the tight turn between the b and c strands of the propeller blade. The innermost strand is the a strand and the outermost is d. Clathrin is without a recognizable sequence repeat and does not contain the typical WD repeat pattern or the highly conserved aspartic acid on the upper surface of the ring. However, given the apparent universal need for at least one very tight turn either between strands a and b or b and c of all known b propeller proteins, the high frequency of small residues in the inner strands of the propeller blades and hydrophobic aromatics in the remaining strands, it is not surprising that all b propeller proteins show some weak sequence similarity. More than 160 different WD repeat proteins have been identified so far, the overwhelming majority of which are found in eukaryotes (4). The occasional WD repeat proteins found in prokaryotes probably arose by horizontal gene transfer, but that family has not expanded there to the extent it has in eukaryotes (for example, there are more than 50 WD repeat proteins in the yeast Saccharomyces cerevisiae). In contrast, the non-WD repeat b propellers frequently are found in prokaryotes. It is not known how many non-WD repeat propellers exist because they do not have a signature sequence that allows for their easy identification. However, more than 15 have been crystallized so far, approximately half from prokaryotes. Some proteins are made up of more than one propeller. For example, hemopexin has two four-bladed propellers per chain and dimerizes to form a symmetrical structure of four four-bladed propellers (9). The individual blades of these propellers can be superimposed on the blades of the Gb subunit and with other non-WD repeat propellers with a fit of 0.9to 2.2-Å rms deviation (10, 11), but the overall structure and degree of symmetry varies among the proteins. For example, the central tunnel can be very circular when the proteins are highly symmetrical or oval, as in clathrin and neuraminidase, when the structure is less symmetrical (2, 9). The reduced symmetry appears to arise most often from the lack of a fourth strand, which sometimes is replaced by a short helix, or from the fact