Amyloid of Rnq1p, the basis of the [ PIN ⁺ ] prion, has a parallel in-register β-sheet structure

Abstract

The [PIN ؉ ] prion, a self-propagating amyloid form of Rnq1p, increases the frequency with which the [PSI ؉ ] or [URE3] prions arise de novo. Like the prion domains of Sup35p and Ure2p, Rnq1p is rich in N and Q residues, but rnq1⌬ strains have no known phenotype except for inability to propagate the [PIN ؉ ] prion. We used solidstate NMR methods to examine amyloid formed in vitro from recombinant Rnq1 prion domain (residues 153-405) labeled with Tyr-1-13 C (14 residues), Leu-1-13 C (7 residues), or Ala-3-13 C (13 residues). The carbonyl chemical shifts indicate that most Tyr and Leu residues are in ␤-sheet conformation. Experiments designed to measure the distance from each labeled residue to the next nearest labeled carbonyl showed that almost all Tyr and Leu carbonyl carbon atoms were Ϸ0.5 nm from the next nearest Tyr and Leu residues, respectively. This result indicates that the Rnq1 prion domain forms amyloid consisting of parallel ␤-strands that are either in register or are at most one amino acid out of register. Similar experiments with Ala-3-13 C indicate that the ␤-strands are indeed in-register. The parallel in-register structure, now demonstrated for each of the yeast prions, explains the faithful templating of prion strains, and suggests as well a mechanism for the rare hetero-priming that is [PIN ؉ ]'s defining characteristic.T hree genetic criteria designed to identify infectious proteins [PIN ϩ ] has all of the genetic properties of a prion (4, 5), and was shown to be a self-propagating amyloid form of Rnq1p (4, 6), a protein rich in asparagine (N) and glutamine (Q), which had been shown to form self-propagating aggregates in vivo (7). Deletion of the RNQ1 gene produces no evident phenotype (7) (14), it is likely that variants of [PIN ϩ ] are different amyloid structures, although the precise nature of such differences remains unknown for any system at present.Aggregates formed in vitro from Rnq1p are filamentous and stain with thioflavin T (7). Circular dichroism indicates that these aggregates are high in ␤-sheet content and, on exposure to proteinase K, show partial resistance (6). These studies indicate that the Rnq1p aggregates are amyloid fibers. Moreover, these fibers formed in vitro from recombinant Rnq1p are infectious for yeast spheroplasts, efficiently transmitting the [PIN ϩ ] gene (6).Because amyloid is neither soluble nor crystalline, neither solution NMR nor x-ray crystallography is suitable for elucidating its detailed structure, although the study of amyloid-like microcrystals of short oligopeptides has revealed details of packing and sidechain interactions that are relevant to amyloid structures (15,16). Electron spin resonance studies have been used to study amylin (IAPP) and ␣-synuclein (17, 18) as well as amyloid of PrP (19). Solid-state NMR has been particularly useful for the elucidation of the structure of amyloids (reviewed in ref. 20). Selective labeling of particular amino acid residues with magnetic nuclear isotopes 13 C and 15 N, combined with pulse sequences designed...

Functionally Redundant Isoforms of a Yeast Hsp70 Chaperone Subfamily Have Different Antiprion Effects

“…It is also possible that specific Hsp70/40 pairings might alter prions independently of Hsp104, either directly or through effects on as yet unidentified cellular processes important for prion propagation or that the different Hsp70 isoforms differentially alter expression of other factors that influence prions. Although the fungal prions analyzed to date all have parallel in-register b-sheet structure, different prions, or even strains of the same prions, have different overall conformations and dynamics of growth and replication (Kushnirov and Ter-Avanesyan 1998;Shewmaker et al 2006;Tanaka et al 2006;Baxa et al 2007;Wickner et al 2008). Such conformational differences are likely a basis for differences in how well the chaperone machinery interacts with the different amyloids in vivo and how efficiently it is able to influence dynamics of growth and replication required for prion propagation.…”

Amyloid-like Fibrils from a Domain-swapping Protein Feature a Parallel, in-Register Conformation without Native-like Interactions

“…A series of two-dimensional 13 C-13 C NMR spectra with 23-, 54-, and 100-ms PARIS mixing were used to probe longer distance 13 C- 13 C contacts (at 17-kHz MAS and 800-MHz 1 H frequencies). To probe the inter-strand register, PITHIRDS-CT measurements (41,42) were carried out using fibrils prepared from Tyr-1-13 CO-labeled dsGB1 at 600 MHz ( 1 H frequency) and an MAS frequency of 20 kHz, with up to 38.4 ms of total dipolar recoupling. Two fibril samples were prepared: one entirely from Tyr-1- 13 CO labeled protein and the other from the same labeled protein diluted 1:2 with unlabeled protein.…”

The repeat domain of the melanosome fibril protein Pmel17 forms the amyloid core promoting melanin synthesis

“…Using solid-state NMR, amyloids of the Alzheimer's A␤ peptide and the diabetes-associated amyloid peptide amylin are inregister parallel ␤-sheets (identical residues of different molecules aligned along the fiber long axis) (20)(21)(22) (23)(24)(25). Electron spin resonance studies showed in-register parallel ␤-sheet structures of amylin (26), ␣-synuclein (Parkinson's disease) (27), and Tau (Alzheimer's disease and other 'tauopathies') (28).…”