title: Flexibility of the Ure2 prion domain is important for amyloid fibril formation.
abstract: Ure2, the protein determinant of the Saccharomyces cerevisiae prion [URE3], has a natively disordered N-terminal domain that is important for prion formation in vivo and amyloid formation in vitro; the globular C-domain has a glutathione transferase-like fold. In the present study, we swapped the position of the N- and C-terminal regions, with or without an intervening peptide linker, to create the Ure2 variants CLN-Ure2 and CN-Ure2 respectively. The native structural content and stability of the variants were the same as wild-type Ure2, as indicated by enzymatic activity, far-UV CD analysis and equilibrium denaturation. CLN-Ure2 was able to form amyloid-like fibrils, but with a significantly longer lag time than wild-type Ure2; and the two proteins were unable to cross-seed. Under the same conditions, CN-Ure2 showed limited ability to form fibrils, but this was improved after addition of 0.03 M guanidinium chloride. As for wild-type Ure2, allosteric enzyme activity was observed in fibrils of CLN-Ure2 and CN-Ure2, consistent with retention of the native-like dimeric structure of the C-domains within the fibrils. Proteolytically digested fibrils of CLN-Ure2 and CN-Ure2 showed the same residual fibril core morphology as wild-type Ure2. The results suggest that the position of the prion domain affects the ability of Ure2 to form fibrils primarily due to effects on its flexibility.
authors.affiliation: National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, 15 Datun Road, Chaoyang District, Beijing, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Yu
authors.forename: Hai-Yan
authors.initials: HY
authors.lastname: Wang
authors.forename: Ming
authors.initials: M
authors.lastname: Bai
authors.forename: Sarah
authors.initials: S
authors.lastname: Perrett
mesh_terms: D000682:Amyloid / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal / Q000502:physiology; D005979:Glutathione Peroxidase / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D009154:Mutation; D011328:Prions / Q000235:genetics / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000682:Amyloid; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae