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

Abstract

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-1-13 C 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 inregister 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.[PSI+] prion | solid-state NMR | dipolar recoupling | amyloid | Sup35 P rions are infectious proteins, mostly self-propagating amyloids. Amyloid is a filamentous polymer, rich in β-sheet structure, in which the β-strands run perpendicular to the long axis of the filament and the hydrogen bonds joining β-strands to make a sheet are along the long axis of the filament. In mammals, prions are uniformly lethal diseases, caused by amyloid formation of the PrP protein. In yeast and fungi, prions are not uniformly fatal and have widely varying effects (reviewed in ref. 1). Perhaps the most remarkable feature of prions is that they have strains or variants, distinct self-propagating forms of the same protein, analogous to alleles of a gene, each relatively stably propagated. The existence of different self-propagating prion variants implies an array of self-propagating structures, each based on the same protein sequence. Because each prion variant is self-propagating, and each variant represents a different amyloid structure/ conformation, there must be some mechanism by which the protein can template its conformation. This mechanism must operate for each of the many conformations that are possible for a given prion.An amino acid residue in a β-sheet can have interactions in three dimensions (Fig. 1A): (a) along the peptide chain; (b) with the residues it faces within the β-sheet but perpendicular to the peptide chain, including the residues to which its main chain N-H and >C = O are hydrog...

Combining DNP NMR with segmental and specific labeling to study a yeast prion protein strain that is not parallel in-register

“…relaxation rates for many amino acids in NM are consistent with the 4.8-Å spacing for between beta-strands (12,17,18). However, the PITHIRDS-CT does not report on the chemical identity of the other isotopically labeled site, and the extracted distances are very sensitive to how the geometry of neighboring spins is modeled (16).…”

“…1). To do so, we prepared chimeric His 6 -NM[ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14) ]-Npu[ (15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25) ]-His 6 , expressed in natural-abundance media (Fig. 1C).…”

“…Five of these alanines are near residues interpreted to be in a parallel in-register quaternary structure based upon relaxation rates determined from PITHIRDS-CT experiments (Fig. 4B) (18). We first prepared an NM fiber sample where alanine residues were isotopically enriched at both the Cα and N positions.…”

On the lack of polymorphism in Aβ‐peptide aggregates derived from patient brains

“…lateral packing) hydrophobic interactions of hydrophobic residues. 24 We first measure the intramolecular interaction through hydrophobic contacts between phenylalanine (F 19 ) and leucine (L 34 ) in adjacent b-sheets of the strand-loop-strand units (Fig. 1).…”

“…Examples are the contacts between residues R 5 and V 24 of the adjacent of strand-loop-strand units in in vivo three-fold, H 13 and V 40 of the in vitro three-fold, and H 13 and V 40 within the strand-loop-strand units of the in vitro two-fold. The contact distances in Table III shows in the six-layer in vivo model with three-fold that the Ca-Ca distance measured between residues R 5 and V 24 grows from an initial 10 to 26 Å averaged over the last 100 ns. Hence, side-chain packing between R 5 and V 24 is not sufficient to conserve the Ushaped motifs.…”