Prion amyloid structure explains templating: how proteins can be genes

Abstract

The yeast and fungal prions determine heritable and infectious traits, and are thus genes composed of protein. Most prions are inactive forms of a normal protein as it forms a self-propagating filamentous β-sheet-rich polymer structure called amyloid. Remarkably, a single prion protein sequence can form two or more faithfully inherited prion variants, in effect alleles of these genes. What protein structure explains this protein-based inheritance? Using solid-state nuclear magnetic resonance, we showed that the infectious amyloids of the prion domains of Ure2p, Sup35p and Rnq1p have an in-register parallel architecture. This structure explains how the amyloid filament ends can template the structure of a new protein as it joins the filament. The yeast prions [PSI(+)] and [URE3] are not found in wild strains, indicating that they are a disadvantage to the cell. Moreover, the prion domains of Ure2p and Sup35p have functions unrelated to prion formation, indicating that these domains are not present for the purpose of forming prions. Indeed, prion-forming ability is not conserved, even within Saccharomyces cerevisiae, suggesting that the rare formation of prions is a disease. The prion domain sequences generally vary more rapidly in evolution than does the remainder of the molecule, producing a barrier to prion transmission, perhaps selected in evolution by this protection.

Fig. 1

One protein sequence can produce several heritable prion variants. How can a protein structure be self-propagating? We have suggested that the in-register parallel β-sheet architecture, compatible with many different structures, is uniquely able to explain protein structure templating.

Fig. 2

Yeast and fungal prions. The most widely studied prions of S. cerevisiae and Podospora anserina are diagramed. For the yeast prion proteins Ure2p and Sup35p, prion amyloid formation prevents normal function, thus producing a phenotype. The normal function of Rnq1p is unknown. The normal function of the HET-s protein is its prion function.

Fig. 3

A. The four types of β-sheet architecture. Only the in-register parallel form results in a labeled atom in a residue of one molecule being about 4.7 angstroms from the same atom of the same residue of an adjacent molecule. The solid-state NMR experiments measure this distance. The large dot represents a single amino acid residue labeled with ¹³C at its carbonyl carbon. B. If a prion domain can be shuffled and still be a prion, it is likely to have an in-register parallel architecture. The specificity of prion propagation requires that interacting residues have some relation to each other. If the architecture is antiparallel, β helix or out of register parallel, that relation would have to be one of non-identity in most cases. Shuffling the sequence would disrupt this relation. However, in the case of a parallel in-register β sheet, it is identical residues whose side-chains interact to determine the sequence-specific prion propagation. Shuffling the sequence does not prevent this same interaction, although the residues will be in a different sequence, identical residues can still interact to produce an amyloid filament.

Fig. 4

Solid-state NMR data demonstrates the in-register parallel architecture for the yeast prion amyloids - the prion domain of Rnq1 in this case (Wickner, et al., 2008). The rapid decay of the NMR signal for 1-¹³C-Tyr or 1-¹³C-Leu labeled molecules indicates that these atoms are ~5 angstroms from their nears labeled neighbor (B). To show that that nearest neighbor is in a different molecule, labeled molecules are diluted with unlabeled molecules and the observed results (B, inverted blue triangles) are in accord with the expected results (inverted empty green triangles) as diagrammed in (A) for the nearest neighbor being in a different molecule. To determine if the structure is really in-register, methyl-¹³C-Ala labeled molecules were examined. Even a single residue out of register would result in a slow signal decay (C). The rapid decay confirms the in-register parallel architecture (D). Similar results have been obtained for the prion domains of Sup35p (Shewmaker, et al., 2006) and Ure2p (Baxa, et al., 2007).

Fig. 5

In-register parallel structure explains the ability of each yeast prion amyloid to faithfully template any of several "variant" structures. We suggest that variants differ in the locations of the turns (the folds of the sheet). Side chain–side chain interactions along the filament axis enforce the same locations for turns in the molecule newly joining the end of the filament as those in the previous molecule. The black dots represent a particular residue, say Gln46, in a prion domain sequence.