The role of helix 1 aspartates and salt bridges in the stability and conversion of prion protein.
A key event in the pathogenesis of transmissible spongiform encephalopathies is the conversion of PrP-sen to PrP-res. Morrissey and Shakhnovich (Morrissey, M. P., and Shakhnovich, E. I. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 11293-11298) proposed that the conversion mechanism involves critical interactions at helix 1 (residues 144-153) and that the helix is stabilized on PrP-sen by intra-helix salt bridges between two aspartic acid-arginine ion pairs at positions 144 and 148 and at 147 and 151, respectively. Mutants of the hamster prion protein were constructed by replacing the aspartic acids with either asparagines or alanines to destabilize the proposed helix 1 salt bridges. Thermal and chemical denaturation experiments using circular dichroism spectroscopy indicated the overall structures of the mutants are not substantially destabilized but appear to unfold differently. Cell-free conversion reactions performed using ionic denaturants, detergents, and salts (conditions unfavorable to salt bridge formation) showed no significant differences between conversion efficiencies of mutant and wild type proteins. Using conditions more favorable to salt bridge formation, the mutant proteins converted with up to 4-fold higher efficiency than the wild type protein. Thus, although spectroscopic data indicate the salt bridges do not substantially stabilize PrP-sen, the cell-free conversion data suggest that Asp-144 and Asp-147 and their respective salt bridges stabilize PrP-sen from converting to PrP-res.
Speare
Jonathan O
JO
Laboratory of Persistent Viral Diseases, Rocky Mountain Laboratories, NIAID, National Institutes of Health, Hamilton, Montana 59840, USA.
Rush
Thomas S
TS
Bloom
Marshall E
ME
Caughey
Byron
B
D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry; D002474:Cell-Free System; D002942:Circular Dichroism; D006224:Cricetinae; D004220:Disulfides / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006358:Hot Temperature; D008647:Mesocricetus; D016296:Mutagenesis; D011328:Prions / Q000737:chemistry* / Q000235:genetics*; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012492:Salts / Q000737:chemistry*
D004220:Disulfides; D011328:Prions; D012492:Salts; D001224:Aspartic Acid; D019791:Guanidine