title: The core of Ure2p prion fibrils is formed by the N-terminal segment in a parallel cross-β structure: evidence from solid-state NMR.
abstract: Intracellular fibril formation by Ure2p produces the non-Mendelian genetic element [URE3] in Saccharomyces cerevisiae, making Ure2p a prion protein. We show that solid-state NMR spectra of full-length Ure2p fibrils, seeded with infectious prions from a specific [URE3] strain and labeled with uniformly (15)N-(13)C-enriched Ile, include strong, sharp signals from Ile residues in the globular C-terminal domain (CTD) with both helical and nonhelical (13)C chemical shifts. Treatment with proteinase K eliminates these CTD signals, leaving only nonhelical signals from the Gln-rich and Asn-rich N-terminal segment, which are also observed in the solid-state NMR spectra of Ile-labeled fibrils formed by residues 1-89 of Ure2p. Thus, the N-terminal segment, or "prion domain" (PD), forms the fibril core, while CTD units are located outside the core. We additionally show that, after proteinase K treatment, Ile-labeled Ure2p fibrils formed without prion seeding exhibit a broader set of solid-state NMR signals than do prion-seeded fibrils, consistent with the idea that structural variations within the PD core account for prion strains. Measurements of (13)C-(13)C magnetic dipole-dipole couplings among (13)C-labeled Ile carbonyl sites in full-length Ure2p fibrils support an in-register parallel β-sheet structure for the PD core of Ure2p fibrils. Finally, we show that a model in which CTD units are attached rigidly to the parallel β-sheet core is consistent with steric constraints.
authors.affiliation: Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Dmitry S
authors.initials: DS
authors.lastname: Kryndushkin
authors.forename: Reed B
authors.initials: RB
authors.lastname: Wickner
authors.forename: Robert
authors.initials: R
authors.lastname: Tycko
mesh_terms: D000682:Amyloid / Q000737:chemistry*; D005979:Glutathione Peroxidase / Q000737:chemistry*; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D011328:Prions / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000682:Amyloid; D011328:Prions; D029701:Saccharomyces cerevisiae Proteins; D005979:Glutathione Peroxidase; C067020:URE2 protein, S cerevisiae