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Characterization of β-Sheet Structure in Ure2p1-89 Yeast Prion Fibrils by Solid-State Nuclear Magnetic Resonance

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posted on 13.11.2007, 00:00 by Ulrich Baxa, Reed B. Wickner, Alasdair C. Steven, D. Eric Anderson, Lyuben N. Marekov, Wai-Ming Yau, Robert Tycko
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 Ure2p1-89 in vitro. Data include measurements of intermolecular magnetic dipole−dipole couplings in samples that are 13C-labeled at specific sites and two-dimensional 15N−13C and 13C−13C NMR spectra of samples that are uniformly 15N- and 13C-labeled. Intermolecular dipole−dipole couplings indicate that the β-sheets in Ure2p1-89 fibrils have an in-register parallel structure. An in-register parallel β-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 Ure2p1-89 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., Riek, 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 Ure2p1-89 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 Ure2p1-89 fibrils suggests that certain portions of the amino acid sequence may not participate in a rigid β-sheet structure, possibly including portions of the Asn-rich segment between residues 44 and 76.

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