Folding a WD repeat propeller. Role of highly conserved aspartic acid residues in the G protein beta subunit and Sec13.
The beta subunit of the heterotrimeric G proteins that transduce signals across the plasma membrane is made up of an amino-terminal alpha-helical segment followed by seven repeating units called WD (Trp-Asp) repeats that occur in about 140 different proteins. The seven WD repeats in Gbeta, the only WD repeat protein whose crystal structure is known, form seven antiparallel beta sheets making up the blades of a toroidal propeller structure (Wall, M. A., Coleman, D. E., Lee, E., Iniguez-Lluhi, J. A., Posner, B. A., Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047-1058; Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 369-374). It is likely that all proteins with WD repeats form a propeller structure. Alignment of the sequence of 918 unique WD repeats reveals that 85% of the repeats have an aspartic acid (D) residue (not the D of WD) in the turn connecting beta strands b and c of each putative propeller blade. We mutated each of these conserved Asp residues to Gly individually and in pairs in Gbeta and in Sec13, a yeast WD repeat protein involved in vesicular traffic, and then analyzed the ability of the mutant proteins to fold in vitro and in COS-7 cells. In vitro, most single mutant Gbeta subunits fold into Gbetagamma dimers more slowly than wild type to a degree that varies with the blade. In contrast, all single mutants form normal amounts of Gbetagamma in COS-7 cells, although some dimers show subtle local distortions of structure. Most double mutants assemble poorly in both systems. We conclude that the conserved Asp residues are not equivalent and not all are essential for the folding of the propeller structure. Some may affect the folding pathway or the affinity for chaperonins. Mutations of the conserved Asp in Sec13 affect folding equally in vitro and in COS-7 cells. The repeats that most affected folding were not at the same position in Sec13 and Gbeta. Our finding, both in Gbeta and in Sec13, that no mutation of the conserved Asp entirely prevents folding suggests that there is no obligatory folding order for each repeat and that the folding order is probably not the same for different WD repeat proteins, or even necessarily constant for the same protein.
Garcia-Higuera
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Cardiovascular Division, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Gaitatzes
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C
Smith
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Neer
E J
EJ
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid; D019556:COS Cells; D003198:Computer Simulation; D017124:Conserved Sequence; D019281:Dimerization; D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry*; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry*; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D028861:Nuclear Pore Complex Proteins; D017354:Point Mutation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D014162:Transfection; D014364:Tryptophan
D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D028861:Nuclear Pore Complex Proteins; D011994:Recombinant Proteins; C079620:SEC13 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D014364:Tryptophan; D019204:GTP-Binding Proteins