Locating folds of the in-register parallel β-sheet of the Sup35p prion domain infectious amyloid

• Published in: Proceedings of the National Academy of Sciences - PNAS 2014-10, Vol.111 (43), p.E4615-E4622
• Main Authors: Gorkovskiy, Anton, Thurber, Kent R, Tycko, Robert, Wickner, Reed B
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amyloid - chemistry; Biological Sciences; Carbon-13 Magnetic Resonance Spectroscopy; Molecular Sequence Data; Mutant Proteins - chemistry; Peptide Termination Factors - chemistry; PNAS Plus; Prions - chemistry; Protein Structure, Secondary; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Transfection
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.1417974111

Significance Infectious proteins (prions) are capable of encoding genetic information by templating their conformation, just as DNA templates its sequence. The mechanism of this templating has not been clear. We provide definitive proof that the architecture of amyloid of the prion domain of yeast prion protein Sup35p is a folded in-register parallel β-sheet, and our data identify some of the sites of folds in the sheet. This architecture naturally suggests a templating mechanism based on favorable interactions among aligned side chains of identical amino acids. This is the only mechanism suggested to date for such a conformation templating. The [PSI+] prion is a self-propagating amyloid of the translation termination factor, Sup35p, of Saccharomyces cerevisiae . The N-terminal 253 residues (NM) of this 685-residue protein normally function in regulating mRNA turnover but spontaneously form infectious amyloid in vitro. We converted the three Ile residues in Sup35NM to Leu and then replaced 16 single residues with Ile, one by one, and prepared Ile-1- ¹³C amyloid of each mutant, seeding with amyloid formed by the reference sequence Sup35NM. Using solid-state NMR, we showed that 10 of the residues examined, including six between residues 30 and 90, showed the ∼0.5-nm distance between labels diagnostic of the in-register parallel amyloid architecture. The five scattered N domain residues with wider spacing may be in turns or loops; one is a control at the C terminus of M. All mutants, except Q56I, showed little or no [PSI+] transmission barrier from the reference sequence, suggesting that they could assume a similar amyloid architecture in vitro when seeded with filaments of reference sequence Sup35NM. Infection of yeast cells expressing the reference SUP35 gene sequence with amyloid of several mutants produced [PSI+] transfectants with similar efficiency as did reference sequence Sup35NM amyloid. Our work provides a stringent demonstration that the Sup35 prion domain has the folded in-register parallel β-sheet architecture and suggests common locations of the folds. This architecture naturally suggests a mechanism of inheritance of conformation, the central mystery of prions.