The hydrophobin EAS from the fungus Neurospora crassa forms functional amyloid fibrils called rodlets that facilitate spore formation and dispersal. Self-assembly of EAS into fibrillar rodlets occurs spontaneously at hydrophobic:hydrophilic interfaces and the rodlets further associate laterally to form amphipathic monolayers. We have used site-directed mutagenesis and peptide experiments to identify the region of EAS that drives intermolecular association and formation of the cross-β rodlet structure. Transplanting this region into a nonamyloidogenic hydrophobin enables it to form rodlets. We have also determined the structure and dynamics of an EAS variant with reduced rodlet-forming ability. Taken together, these data allow us to pinpoint the conformational changes that take place when hydrophobins self-assemble at an interface and to propose a model for the amphipathic EAS rodlet structure.A myloid fibrils were first identified in association with human diseases, but recent discoveries show that the amyloid ultrastructure also contributes to important functions in normal biology (1, 2). In bacteria, fungi, insects, fish, and mammals, amyloid structures perform a wide variety of roles (3). Functional amyloids in the form of fibrillar rodlets composed of class I hydrophobin proteins are found in filamentous fungi. These hydrophobins are small proteins that are secreted as monomers and self-assemble into rodlets that pack to form amphipathic monolayers at hydrophilic: hydrophobic boundaries, such as the surface of the growth medium (4). These proteins are extremely surface active and lower the surface tension of the aqueous growth medium, allowing hyphae to break through the surface and to produce aerial structures (5, 6). Many of these aerial structures subsequently become coated with amyloid rodlets, creating a hydrophobic layer that serves multiple purposes, including conferring water resistance to spores for easier dispersal in air (7), preventing wetting or collapse of gas transfer channels (8), enhancing adherence to waxy surfaces such as leaves during infection of rice plants by Magnaporthe grisea (9), and mediating evasion of the immune system as is observed in Aspergillus fumigatus infections (10).Hydrophobins are characterized by the presence of eight cysteine residues that form four disulphide bonds, but the hydrophobin family can be further divided into two classes based on the spacing of the conserved cysteine residues and the nature of the amphipathic monolayers that they form (11). Class I, but not class II, hydrophobins form amyloid-like rodlets that are extremely robust and require treatment with strong acid to induce depolymerization. The amphipathic monolayers formed by class II hydrophobins are not fibrillar and can be dissociated by treatment with detergent and alcohol solutions. The soluble, monomeric forms of hydrophobins share a unique β-barrel topology, and all have a relatively large exposed hydrophobic area on the protein monomer surface (4). The diversity in sequence and chain length ...

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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.Class I hydrophobins are a family of small amphipathic proteins that are produced by filamentous fungi in a monomeric form but are able to self-assemble into amphipathic monolayers composed of amyloid-like structures known as rodlets (1-3). The polymerization of the hydrophobins occurs on contact with a hydrophobic:hydrophilic interface, such as an air:water boundary or when hydrophobins are secreted from the spores and come into contact with the air. Members of the hydrophobin family are characterized by the presence of four disulfide bonds. The amphipathic nature of hydrophobins drives them to the surface of solutions, where they reduce the surface tension (1).Some of the functional roles of the class I hydrophobins include acting as a surfactant at the air:water boundary to reduce the surface tension, which is a barrier to aerial growth of hyphae, and also to form a robust protein coat on spores. This coating provides a hydrophobic external surface that resists wetting and thus facilitates spore dispersal in air (4 -6). The class I hydrophobin rodlets share many of the structural characteristics of amyloid fibrils; formation of the insoluble, fibrillar rodlets is accompanied by conformational change to an ordered cross-␤-secondary structure form and the polymerized rodlets, but not the monomeric form of the protein, bind to the dye thioflavin T (ThT) 4 (2, 7, 8). However, in contrast to other amyloid fibrils, which can often be solubilized by treatment with denaturants such as guanidine hydrochloride or solvents such as dimethyl sulfoxide (9), treatment with acids such as formic and TFA has been reported to be the only method capable of depolymerizing hydrophobin rodlets and regenerating the monomeric form of the hydrophobin proteins in solution (10,11). This is similar to the curli and tafi fibrils produced by bacteria, which have been shown to be functional amyloid and which also req...

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