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J. Biol. Chem., Vol. 280, Issue 44, 37149-37158, November 4, 2005

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Structure of the Prion Ure2p in Protein Fibrils Assembled in Vitro^*

From the Laboratoire d'Enzymologie et Biochimie Structurales, CNRS, Avenue de la Terrasse, 91198 Gif-sur-Yvette Cedex, France

The Ure2 protein from the yeast Saccharomyces cerevisiae has prion properties. In vitro and at neutral pH, soluble Ure2p spontaneously forms long, straight, insoluble protein fibrils. Two models have been proposed to account for the assembly of Ure2p into protein fibrils. The "amyloid backbone" model postulates that a segment ranging from 40 to 70 amino acids in the flexible N-terminal domain from different Ure2p molecules forms a parallel superpleated -structure running along the fibrils (Kajava, A. V., Baxa, U., Wickner, R. B., and Steven, A. C. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 7885-7890). The second model hypothesizes that assembly of full-length Ure2p is driven by limited conformational rearrangements and non-native inter- and/or intramolecular interactions between Ure2p monomers (Bousset, L., Thomson, N. H., Radford, S. E., and Melki, R. (2002) EMBO J. 21, 2903-2911). Here, we performed a cysteine scan on residues located in the N- and C-terminal parts of Ure2p to determine whether these domains interact. Amino acid sequences centered around residue 6 in the N-terminal domain of Ure2p and residue 137 in the C-terminal moiety interacted at least transiently via intramolecular interactions. We documented the assembly properties of a Ure2p variant in which a disulfide bond was established between the N- and C-terminal domains and showed that it possesses assembly properties indistinguishable from those of wild-type Ure2p. We probed the structure of Ure2pC6C137 within the fibrils and demonstrate that the polypeptide is in a conformation similar to that of its soluble assembly-competent state. Our results constitute the first structural characterization of the N-terminal domain of Ure2p in both its soluble assembly-competent and fibrillar forms. Our data indicate that the flexibility of the N-terminal domain and conformational changes within this domain are essential for fibril formation and provide new insight into the conformational rearrangements that lead to the assembly of Ure2p into fibrils and the propagation of the [URE3] phenotype in yeast.

Received for publication, June 27, 2005 , and in revised form, August 9, 2005.

^* This work was supported by the French Ministry of Education, Research, and Technology through the Groupement d'Intérêt Scientifique Prion, by CNRS, and by INSERM. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-3.

¹ Both authors contributed equally to this work.

³ Present address: EMBL, 38042 Grenoble Cedex 9, France.

² To whom correspondence may be addressed. Tel.: 33-169-823-486; Fax: 33-169-823-129; E-mail: redeker{at}lebs.cnrs-gif.fr.

⁴ To whom correspondence may be addressed. Tel.: 33-169-823-503; Fax: 33-169-823-129; E-mail: melki{at}lebs.cnrs-gif.fr.

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