Recruitment of class I hydrophobins to the air:water interface initiates a multi-step process of functional amyloid formation

Abstract

Class I fungal hydrophobins form amphipathic monolayers composed of amyloid rodlets. This is a remarkable case of functional amyloid formation in that a hydrophobic:hydrophilic interface is required to trigger the self-assembly of the proteins. The mechanism of rodlet formation and the role of the interface in this process have not been well understood. Here, we have studied the effect of a range of additives, including ionic liquids, alcohols, and detergents, on rodlet formation by two class I hydrophobins, EAS and DewA. Although the conformation of the hydrophobins in these different solutions is not altered, we observe that the rate of rodlet formation is slowed as the surface tension of the solution is decreased, regardless of the nature of the additive. These results suggest that interface properties are of critical importance for the recruitment, alignment, and structural rearrangement of the amphipathic hydrophobin monomers. This work gives insight into the forces that drive macromolecular assembly of this unique family of proteins and allows us to propose a three-stage model for the interface-driven formation of rodlets.

FIGURE 1.

The hydrophobins DewA and EAS_Δ15 form amphipathic monolayers composed of rodlets. Negatively stained transmission electron micrographs of rodlet monolayers formed by DewA (A) and EAS_Δ15 (B). C, images from a video contact angle device showing the side profile of water droplets placed on OTS-treated silicon wafers that had been precoated with DewA or EAS_Δ15 proteins or water.

FIGURE 2.

Ionic liquids inhibit the extent of hydrophobin self-assembly into amyloid-like rodlets. Solutions containing 10 μm DewA or 4.3 μm EAS_Δ15, 32 μm ThT, and ionic liquid solutions were agitated at 3000 rpm for varying lengths of time. A, extent of DewA rodlet formation in water, 25% EaN, and 25% TeaAc. B, extent of DewA rodlet formation in varying concentrations of EaN. C, extent of EAS_Δ15 rodlet formation in varying concentrations of EaN. Error bars shown are one S.D. from the mean of three replicates. Lines are drawn only to guide the eye.

FIGURE 3.

Alcohols reduce the extent of rodlet formation in a manner consistent with the reduction in surface tension. Solutions containing hydrophobin, ThT, and various alcohols were agitated at 3000 rpm for varying lengths of time. A, EAS_Δ15 + ethanol; B, EAS_Δ15 + 1-propanol; C, EAS_Δ15 + methanol; and D, DewA + ethanol. Error bars shown are one S.D. from the mean of three replicates. E, graph showing the relationship between surface tension and varying concentration of ethanol, methanol, and 1-propanol in water using published data (37). Arrows and gray shading indicate the approximate surface tension corresponding to alcohol concentrations above which no EAS_Δ15 or DewA rodlet formation was observed in a period up to 10× T1, where T1 is the time constant for the rate of self-assembly of the corresponding protein in water.

FIGURE 4.

Detergent reduces the extent of rodlet formation in a manner consistent with the reduction in surface tension. Solutions containing hydrophobin, ThT, and Triton X-100 were agitated at 3000 rpm for varying lengths of time. A, EAS_Δ15; B, DewA. Error bars shown are one S.D. from the mean of three replicates.

FIGURE 5.

Ionic liquids and alcohols do not dramatically perturb the structure of the hydrophobins in solution, even under conditions where rodlet formation is affected significantly. A, far UV CD spectra of DewA in water and in 33% EaN, 33% TeaAc, and 50% TeaAc. B, far UV CD spectra of EAS_Δ15 in water and in 10% ethanol, 3% 1-propanol, and 13% methanol (all v/v). C, ¹⁵N-HSQC spectra of ethanol titration into EAS_Δ15 (in 20 mm sodium phosphate containing 10% (v/v) D₂O and 2 μm 2,2-dimethyl-2-silapentanesulfonic acid, pH 6.0). Resonance assignments (BioMagResBank code 15863) are indicated beside each signal. mdeg, millidegrees.

FIGURE 6.

Model of hydrophobin assembly at the interface and its inhibition by additives. A, schematic diagram showing the possible effect of additives on hydrophobin assembly. B, proposed sequence of events that leads to rodlet assembly upon hydrophobins encountering an air:water interface. Hydrophobin monomers are represented by gray/purple ovals with gray and purple patches corresponding to hydrophobic and hydrophilic regions, respectively. Disordered loop regions are represented by gray curves. Additives are illustrated as black dots. Concentration and reorientation of hydrophobins at the air:water interface are required for conformational change to take place. This conformational change then allows adjacent hydrophobin monomers to strongly interact with each other and eventually results in rodlet formation. Additives may interfere with this process by binding to the air:water and hydrophobin:water interfaces.