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It is now possible to identify over 30 functional subfamilies among the WD-repeat-containing proteins found in the completed genomes. The majority of these subfamilies have at least one member for which experimental data allow assignment to a cellular pathway or process. Half of the 63 WD-repeat-containing proteins in Saccharomyces cerevisiae, half of the 70 in Caenorhabditis elegans, and a third of the 100 plus predicted in Drosophila can be assigned to 23 of these functional subfamilies. Perhaps indicative of the future, 33 WD-repeat-containing proteins from the partial genome of Arabidopsis thaliana can now be assigned to 18 of these subfamilies. These assignments have been made possible by combining traditional sequence similarity with an implied common beta propeller structural context to obtain measures of protein-protein surface similarity. The beta propeller structural context is represented in the form of a Hidden Markov Model. The procedure is completely automated.

The ␤ subunit of the heterotrimeric G proteins that transduce signals across the plasma membrane is made up of two distinct regions as follows: an amino-terminal ␣-helical segment, followed by 7 repeating units called WD repeats that occur in about 140 different proteins (reviewed in Refs. 1 and 2). Members of the family of WD repeat proteins do not have an immediately obvious common function but are involved in diverse cellular pathways such as signal transduction, pre-mRNA splicing, transcriptional regulation, cytoskeletal assembly, and vesicular traffic (2).Each WD repeat consists of a conserved core of approximately 40 amino acids (typically bracketed by the dipeptides GH (glycine-histidine) and WD (tryptophan-aspartic acid)) and a variable region of 7-11 amino acids (2). G␤ is the only WD repeat protein whose crystal structure is known (3-5). The seven WD repeats in G␤ are arranged in a ring to form a propeller structure with seven blades. Each blade of the propeller consists of a four-stranded antiparallel ␤ sheet oriented so that the outer surfaces of the torus are composed of the sheet edges, whereas the turns protrude from the two flat surfaces (see Fig. 1). It is likely that all proteins with WD repeats form a propeller structure, although with varying numbers of blades corresponding to varying numbers of repeating units. WD repeats are not essential to form a propeller. Other families of proteins with no sequence similarity to WD repeat proteins form propellers whose blades are virtually identical to those in G␤ (reviewed in Ref. 6). Nevertheless, within the subset of propellers formed of WD repeats, it is reasonable to suppose that the most highly conserved residues play an important role either in the function or the structure.The WD repeats are not characterized by a rigidly conserved sequence but rather by their fit to a regular expression that allows limited variation at each position (2). However, alignment of the sequences of 918 unique WD repeats in our data set reveals that one residue is the most conserved; an aspartic acid residue (D, not the D in WD) located in the loop connecting ␤ strands b and c of each propeller blade in G␤ (and presumably in all other WD repeat proteins) occurs in 85% of the repeats. In another 9%, the residue is Glu or Asn. This extraordinary conservation suggests that the Asp residue performs an important function that is shared by all WD repeats. Since the WD repeat proteins do not appear to bind to any common molecule, we tested the hypothesis that the conserved Asp plays a role in the folding of the propeller.The occurrence of a conserved residue at an equivalent position in each repeat allowed us to ask a number of questions. Are all the Asp residues equivalent within a protein? Are the consequences of mutating Asp to Gly the same in different proteins? It is not known whether the WD repeat or other propeller proteins fold by a single or multiple pathways. If there is a single pathway, we would expect that mutation of a critical Asp

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WD repeat proteins are a family of proteins that contain a series of highly conserved internal repeat motifs, usually ending with WD (Trp-Asp). The G beta subunit of heterotrimeric guanine nucleotide binding protein is a member of this family, and its crystal structure has been recently solved at high resolution (Wall et al. (1995) Cell 83, 1047-1058; Sondek et al. (1996) Nature 379, 369-374). Based on the coordinates of G beta, we have constructed a model for the structure of Sec13, a 33 kDa WD repeat protein from Saccharomyces cerevesiae essential for vesicular traffic. The model has been tested using a combination of biophysical and biochemical methods. Sec13 was expressed in Escherichia coli as a hexa-His-tagged protein (H6Sec13) and purified to homogeneity. In contrast to some other WD repeat proteins that are unable to fold into monomeric structures when expressed in E. coli, H6Sec13 was soluble and monomeric in the absence of detergent. The far-UV circular dichroism (CD) spectra of H6Sec13 indicated less than 10% alpha-helix consistent with the model which predicts primarily beta-sheets. H6Sec13 shows a cooperative and irreversible thermal denaturation curve consistent with a tightly packed structure. The CD spectrum shows an unusual positive ellipticity at 229 nm that was attributed to interactions of surface tryptophans since the 229 nm maximum could be abolished by modification of 6.3 +/- 0.3 (n = 3) tryptophans (out of 15 total in the molecule) with N-bromosuccinimide. Our model predicts that three sets of tryptophans are clustered near the surface. As predicted by the model, purified H6Sec13 was completely resistant to trypsin digestion. The concordance of the model of Sec13 presented in this paper with the biochemical and biophysical studies suggests that this model can be useful as a guide to further experiments designed to elucidate the function of Sec13 in vesicular traffic.