Folding of proteins with WD-repeats: comparison of six members of the WD-repeat superfamily to the G protein beta subunit.
The family of WD-repeat proteins comprises over 30 different proteins that share a highly conserved repeating motif [Neer, E. J., Schmidt, C. J., Nambudripad, R., & Smith, T. F. (1994) Nature 371, 297-300]. Members of this family include the signal-transducing G protein beta subunit, as well as other proteins that regulate signal transduction, transcription, pre-mRNA splicing, cytoskeletal organization, and vesicular fusion. The crystal structure of one WD-repeat protein (G beta) has now been solved (Wall et al., 1995; Sondek et al, 1996) and reveals that the seven repeating units form a circular, propeller-like structure with seven blades each made up of four beta strands. It is very likely that all WD-repeat proteins form a similar structure. If so, it will be possible to use information about important surface regions of one family member to predict properties of another. If WD proteins form structures similar to G beta, their hydrodynamic properties should be those of compact, globular proteins, and they should be resistant to cleavage by trypsin. However, the only studied example of a WD-repeat protein, G beta, synthesized in vitro in a rabbit reticulocyte lysate, is unable to fold into a native structure without its partner protein G gamma. The non-WD-repeat amino terminal alpha helix of G beta does not inhibit folding because G beta does not fold even when this region is removed. It is not known whether all WD-repeat proteins are unable to fold when synthesized in an in vitro system. We synthesized seven members of the family in a rabbit reticulocyte lysate, determined their Stokes radius, sedimentation coefficient, and frictional ratio, and assayed their stability to trypsin. Our working definition of folding was that the proteins from globular, trypsin-resistant structures because, except for G beta gamma, their functions are not known or cannot be assayed in reticulocyte lysates. We chose proteins that include amino and carboxyl extensions as well as proteins that are made up entirely of WD-repeats. We show that unlike G beta, several proteins with WD-repeats are able to fold into globular proteins in a rabbit reticulocyte lysate. One protein, beta Trcp, formed large aggregates like G beta, suggesting that it may also require a partner protein. Despite the presence of many potential tryptic cleavage sites, all of the proteins that did fold gave stable large products on tryptic proteolysis, as predicted on the basis of the structure of G beta. These studies suggest that other WD-repeat proteins are likely to form propeller structures similar to G beta.
Garcia-Higuera
I
I
Department of Medicine, Brigham and Women's Hospital, Boston, Massachusetts 02115, USA.
Fenoglio
J
J
Li
Y
Y
Lewis
C
C
Panchenko
M P
MP
Reiner
O
O
Smith
T F
TF
Neer
E J
EJ
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D000595:Amino Acid Sequence; D000818:Animals; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics; D019204:GTP-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics; D008840:Microfilament Proteins / Q000737:chemistry / Q000235:genetics; D008869:Microtubule-Associated Proteins; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011817:Rabbits; D000075370:Receptors for Activated C Kinase; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D029701:Saccharomyces cerevisiae Proteins; D044844:beta-Transducin Repeat-Containing Proteins
C471644:BTRC protein, human; D005656:Fungal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D008869:Microtubule-Associated Proteins; D028861:Nuclear Pore Complex Proteins; D010455:Peptides; D011506:Proteins; D000075370:Receptors for Activated C Kinase; D011994:Recombinant Proteins; C079620:SEC13 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D044844:beta-Transducin Repeat-Containing Proteins; C070301:peptide I; C072061:coronin proteins; D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; C084102:PAFAH1B1 protein, human; D019204:GTP-Binding Proteins