Structure of the Prion Ure2p in Protein Fibrils Assembled in Vitro

Nicolas Fay,Virginie Redeker,Jimmy Savistchenko,Steven Dubois,Luc Bousset,Ronald Melki

doi:10.1074/jbc.m506917200

Nicolas Fay, Virginie Redeker + Show 4 more

https://doi.org/10.1074/jbc.m506917200

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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 beta-structure running along the fibrils. 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. 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.

Highlights

Bakers’ yeast cells exhibiting the non-mendelian genetic element [URE3] (1) show altered negative regulation of nitrogen metabolism (2– 4)
A number of Ure2p variants in which Gly, Ser, Arg, and Phe residues located in the N- and C-terminal parts of the protein were replaced with cysteine residues were generated
A number of Ure2p variants in which Gly, Ser, and Phe residues located in the N- and C-terminal parts of Ure2p were replaced with cysteine residues were generated

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Introduction

Bakers’ yeast cells exhibiting the non-mendelian genetic element [URE3] (1) show altered negative regulation of nitrogen metabolism (2– 4). The prion-promoting activity of region 221–227 suggests an interaction between the N- and C-terminal domains of Ure2p (16). In the latter case, the protein has high aggregation propensity. The simplest explanation for the observation that, when associated, amino acid substitutions in the N- and C-terminal domains of Ure2p increase the rate of [URE3] occurrence is that these substitutions favor a state that has high aggregation propensity, possibly by impairing the interaction between the two domains. A number of Ure2p variants in which Gly, Ser, Arg, and Phe residues located in the N- and C-terminal parts of the protein were replaced with cysteine residues were generated Structure of the Prion Ure2p in Protein Fibrils iologically relevant conditions are built of native-like units in a manner reminiscent of the assembly of other biological polymers (18)

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