ANALYSIS OF THE PHYSICAL-PROPERTIES AND MOLECULAR MODELING OF SEC13 -A WD REPEAT PROTEIN INVOLVED IN VESICULAR TRAFFIC

WD repeat proteins are a family of proteins that contain a series of h ighly conserved internal repeat motifs, usually ending with WD (Trp-As p). The Gp subunit of heterotrimeric guanine nucleotide binding protei n is a member of this family, and its crystal structure has been recen tly solved at high resolution (Wall et al. (1995) Cell 83, 1047-1058 S ondek 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 vesi cular traffic. The model has been tested using a combination of biophy sical and biochemical methods. Sec13 was expressed in Escherichia coli as a hexa-His-tagged protein (H6Sec13) and purified to homogeneity. I n contrast to some other WD repeat proteins that are unable to fold in to monomeric structures when expressed in E. coli, H6Sec13 was soluble and monomeric in the absence of detergent. The far-UV circular dichro ism (CD) spectra of H6Sec13 indicated less than 10% alpha-helix consis tent with the model which predicts primarily beta-sheets. H6Sec13 show s a cooperative and irreversible thermal denaturation curve consistent with a tightly packed structure. The CD spectrum shows an unusual pos itive ellipticity at 229 nm that was attributed to interactions of sur face tryptophans since the 229 nm maximum could be abolished by modifi cation of 6.3 +/- 0.3 (n = 3) tryptophans (out of 15 total in the mole cule) with N-bromosuccinimide. Our model predicts that three sets of t ryptophans are clustered near the surface. As predicted by the model, purified H6Sec13 was completely resistant to trypsin digestion. The co ncordance of the model of Sec13 presented in this paper with the bioch emical and biophysical studies suggests that this model can be useful as a guide to further experiments designed to elucidate the function o f Sec13 in vesicular traffic.