Characterization of β-sheet structure in Ure2p1-89 yeast prion fibrils by solid-state nuclear magnetic resonance

Residues 1-89 constitute the Asn- and Gln-rich segment of the Ure2p protein and produce the [URE3] prion of Saccharomyces cerevisiae by forming the core of intracellular Ure2p amyloid. We report the results of solid-state nuclear magnetic resonance (NMR) measurements that probe the molecular structure of amyloid fibrils formed by Ure2p(1-89), in vitro. Data include measurements of intermolecular magnetic dipole-dipole couplings in samples that are C-13-labeled at specific sites and two-dimensional N-15-C-13 and C-13-C-13 NMR spectra of samples that are uniformly N-15- and C-13-labeled. Intermolecular dipole-dipole couplings indicate that the beta-sheets in Ure2p(1-89) fibrils have an in-register parallel structure. An in-register parallel P-sheet structure permits polar zipper interactions among side chains of Gln and Asn residues and explains the tolerance of [URE3] to scrambling of the sequence in residues 1-89. Two-dimensional NMR spectra of uniformly labeled Ure2p(1-89) fibrils, even when fully hydrated, show NMR linewidths that exceed those in solid-state NMR spectra of fibrils formed by residues 218-289 of the HET-s prion protein of Podospora anserina [as originally reported in Siemer, A. B., Ritter, C., Ernst, M., Rick, R., and Meier, B. H. (2005) Angew. Chem., Int. Ed. 44, 2441-2444 and confirmed by measurements reported here] by factors of three or more, indicating a lower degree of structural order at the molecular level in Ure2p 1 -89 fibrils. The very high degree of structural order in HET-s fibrils indicated by solid-state NMR data is therefore not a universal characteristic of prion proteins, and is likely to be a consequence of the evolved biological function of HET-s in heterokaryon incompatibility. Analysis of cross peak intensities in two-dimensional NMR spectra of uniformly labeled Ure2p(1-89) fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid P-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76.