Mass analysis by scanning transmission electron microscopy and electron diffraction validate predictions of stacked...

Area of Science:

• Biochemistry
• Structural Biology
• Mycology

Background:

• Fungal prions are infectious protein polymers crucial for phenomena like heterokaryon incompatibility.
• The HET-s protein of Podospora anserina possesses a prion domain (HET-s-(218-289)) essential for prion formation and fibril assembly.
• Previous models proposed a specific structure for HET-s prion fibrils involving beta-solenoids.

Purpose of the Study:

• To experimentally validate the proposed structural model of HET-s prion fibrils.
• To investigate the amyloid architecture of HET-s prion fibrils using advanced microscopy techniques.
• To compare the structural organization of HET-s fibrils with those of yeast prion proteins.

Main Methods:

• Electron microscopy (EM) was employed to examine the morphology of HET-s prion fibrils.
• Electron diffraction analysis was performed to determine the structural characteristics, specifically looking for cross-beta structure.
• Scanning transmission electron microscopy (STEM) was used for mass-per-unit-length measurements to assess subunit packing density.

Main Results:

• Electron diffraction confirmed the predicted cross-beta structure with a prominent meridional reflection at (0.47 nm)(-1).
• STEM measurements indicated a packing density of approximately 1.02 subunits per 0.94 nm, consistent with the model's prediction.
• The packing density of HET-s fibrils was found to be half that of yeast prion proteins (Ure2p, Sup35p), suggesting fundamentally different amyloid architectures.

Conclusions:

• The experimental data strongly support the proposed model for HET-s prion fibrils, characterized by the axial stacking of beta-solenoids.
• The study reveals a distinct amyloid architecture for fungal prions like HET-s compared to those found in yeast.
• These findings advance our understanding of prion structure and the diversity of amyloid formation in fungi.