Analysis of the Physical Properties and Molecular Modeling of Sec13: A WD Repeat Protein Involved in Vesicular Traffic

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Abstract
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 Gβ 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 Gβ, 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% α-helix consistent with the model which predicts primarily β-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.