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Fungal prion HET-s as a model for structural complexity and self-propagation in prions

Significance Prions are self-propagating infectious proteins that have been implicated in a number of invariably fatal diseases. Increasing evidence suggests that this self-propagating activity is present in most or all amyloid diseases. To understand the pathology of these diseases, it is imperative to understand the mechanisms involved in self-propagation. To this end, we have used the fungal prion-forming domain HET-s(218–289) as a model system for studying sequence determinants required for structural propagation. We have determined the requirements for folding into the biologically active β-solenoid conformation and characterized the roles of different interactions in the assembly and overall stability of HET-s(218–289) prions. Our results provide insights into the fundamental mechanisms of prion assembly and propagation. The highly ordered and reproducible structure of the fungal prion HET-s makes it an excellent model system for studying the inherent properties of prions, self-propagating infectious proteins that have been implicated in a number of fatal diseases. In particular, the HET-s prion-forming domain readily folds into a relatively complex two-rung β-solenoid amyloid. The faithful self-propagation of this fold involves a diverse array of inter- and intramolecular structural features. These features include a long flexible loop connecting the two rungs, buried polar residues, salt bridges, and asparagine ladders. We have used site-directed mutagenesis and X-ray fiber diffraction to probe the relative importance of these features for the formation of β-solenoid structure, as well as the cumulative effects of multiple mutations. Using fibrillization kinetics and chemical stability assays, we have determined the biophysical effects of our mutations on the assembly and stability of the prion-forming domain. We have found that a diversity of structural features provides a level of redundancy that allows robust folding and stability even in the face of significant sequence alterations and suboptimal environmental conditions. Our findings provide fundamental insights into the structural interactions necessary for self-propagation. Propagation of prion structure seems to require an obligatory level of complexity that may not be reproducible in short peptide models.