The crystal structure of the nitrogen regulation fragment of the yeast prion protein Ure2p

Abstract

The yeast nonchromosomal gene [URE3] is due to a prion form of the nitrogen regulatory protein Ure2p. It is a negative regulator of nitrogen catabolism and acts by inhibiting the transcription factor Gln3p. Ure2p residues 1-80 are necessary for prion generation and propagation. The C-terminal fragment retains nitrogen regulatory activity, albeit somewhat less efficiently than the full-length protein, and it also lowers the frequency of prion generation. The crystal structure of this C-terminal fragment, Ure2p(97-354), at 2.3 Å resolution is described here. It adopts the same fold as the glutathione S-transferase superfamily, consistent with their sequence similarity. However, Ure2p(97-354) lacks a properly positioned catalytic residue that is required for S-transferase activity. Residues within this regulatory fragment that have been indicated by mutational studies to influence prion generation have been mapped onto the three-dimensional structure, and possible implications for prion activity are discussed.

The yeast prion protein Ure2: Structure, function and folding

“…Subsequent availability of crystal structures of the truncated C-domain of Ure2 in both the apo form [28,58] and with glutathione bound [59] has confirmed its classification as a theta (or beta) class GST (see Fig. 3).…”

“…Subsequent biophysical analysis of the purified Ure2 protein has shown that the structural properties of the N-terminal and C-terminal regions are also distinct. The N-terminal domain is extremely sensitive to protease digestion [26][27][28]104] and progressive deletion of the N-terminal domain has no effect on the dimerisation, stability or folding kinetics of the protein in vitro [27,33,35,36,105] (see Fig. 6).…”

“…Ure2 is a 354-amino acid protein consisting of a relatively flexible and protease-sensitive N-terminal region, and a globular C-terminal region [26][27][28]. The C-terminal region of Ure2 shows structural similarity to glutathione transferases (GSTs) and is necessary and sufficient for its regulatory function: Ure2 interacts with the transcription factor Gln3 allowing control of nitrogen catabolite repression, blocking the uptake of poor nitrogen sources in the presence of a good nitrogen source [29,30].…”

Parallel β-Sheets and Polar Zippers in Amyloid Fibrils Formed by Residues 10−39 of the Yeast Prion Protein Ure2p

“…Aggregation of Ure2p into amyloid fibrils is responsible for the self-propagating [URE3] phenotype in S. cerevisiae (47,61,62), in which the cells' ability to stop utilization of ureidosuccinate for pyrimidine biosynthesis in the presence of ammonia is suppressed (63). Residues 97-354 of Ure2p form a globular domain with a glutathione S-transferase fold (64). Residues 1-80 or shorter segments thereof constitute the "prion domain" of Ure2p, responsible for aggregation of the full-length protein (47) and capable of forming amyloid fibrils that can seed fibrillization of the full-length protein (65,66).…”

Production of a Polymer-Forming Fusion Protein inEscerichia coliStrain BL21

“…have at least three cellular components, [URE3], 3,4) [PSI þ ], [5][6][7] and [PIN þ ], [8][9][10] that persist in the cell in the same manner as mammalian prion; hence they are often referred to as yeast prions. The yeast prions are not only attractive research objects in themselves but also useful model experimental systems for elucidating conformational alteration of mammalian prion and polymerforming proteins in general.…”