Characterization of a Basidiomycota hydrophobin reveals the structural basis for a high-similarity Class I subdivision

Abstract

Class I hydrophobins are functional amyloids secreted by fungi. They self-assemble into organized films at interfaces producing structures that include cellular adhesion points and hydrophobic coatings. Here, we present the first structure and solution properties of a unique Class I protein sequence of Basidiomycota origin: the Schizophyllum commune hydrophobin SC16 (hyd1). While the core β-barrel structure and disulphide bridging characteristic of the hydrophobin family are conserved, its surface properties and secondary structure elements are reminiscent of both Class I and II hydrophobins. Sequence analyses of hydrophobins from 215 fungal species suggest this structure is largely applicable to a high-identity Basidiomycota Class I subdivision (IB). To validate this prediction, structural analysis of a comparatively distinct Class IB sequence from a different fungal order, namely the Phanerochaete carnosa PcaHyd1, indicates secondary structure properties similar to that of SC16. Together, these results form an experimental basis for a high-identity Class I subdivision and contribute to our understanding of functional amyloid formation.

Figure 1. SC16 is an active Class I hydrophobin.

(A) Linear dependency of enhanced fluorescence of Thioflavin T induced by aeration and protein concentration. Gentle head over tail overnight aeration on a rotary shaker induced enhanced ThT fluorescence. N = 5 for experiment while N = 3 for control (no shaking and no protein). All error bars indicate standard deviation. (B) AFM image of dried down SC16 in water (aerated) on HOPG reveals SC16 assembled into rodlets. Model prepared using Nanoscope software (version 5.12r3) (C) Width, and (D) length distributions indicates SC16 elongates after initial assembly. The average thickness of the layer is 1.99 ± 0.82 nm.

Figure 2. Solution structure of SC16.

(A) The sequence of SC16 from the first cysteine residue to the C-terminus. The secondary structure is indicated for each residue and the locations of the loops, β-sheets and helix of SC16 are indicated. Cysteine residues are coloured fuchsia and disulphide linkages are indicated by a line. (B) Superposition of the 20 lowest-energy structures of SC16, with only the N, C^α, and C’ atoms shown. (C) A ribbon diagram of the lowest-energy SC16 structure. Disulphide bonds are shown in fuchsia and the β-sheets and loops are indicated.

Figure 3. SC16 structural features are reminiscent of Class II hydrophobins.

(A) Schematics of the secondary structure elements of SC16, the Class I hydrophobins (EAS, DewA and MPG1), and the Class II hydrophobins (HFBI, HFBII, and NC2). Both β-sheets (1-4) and intervening loops (L₁-L₃) are indicated. Superposition of the β-sheet core of SC16 (blue) with the Class I hydrophobins EAS (B; red), DewA (C; green), and MPG1 (D; yellow) and (E) the Class II hydrophobins HFBI, HFBII, and NC2 (purple, teal, and grey, respectively).

Figure 4. Clustering of hydrophobin sequences in the most significant components of a principal component analysis (PCA) of the sequence alignment matrix of 1052 confirmed and predicted hydrophobin sequences.

PFam prediction of Classes is indicated by colour of symbol fill in (A) while phylum is indicated by colour of symbol fill in (B). Hydrophobins whose structures have been resolved are indicated in orange: EAS (Neurospora crassa), MPG1 (Magnaporthe grisea), DewA (Aspergillus nidulans), NC2 (Neurospora crassa), HFBI and HFBII (Trichoderma reesei). Hydrophobins whose structures remain unknown but have been characterized at various levels are indicated in green: SC3 (Schizophyllum commune), VMH2 (Pleurotus ostreatus), RodA (Aspergillus fumigatus) FcHyd5P (Fusarium culmorum). (C) Consensus sequences generated from the alignments of the sequences located within the 14 equal-sized pink rectangles in (A).