Self-assembly of functional, amphipathic amyloid monolayers by the fungal hydrophobin EAS

Abstract

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 ...

Amyloid-based nanosensors and nanodevices

“…[21][22][23][24] Since amyloids are generally derived from natural proteins, they have a low immunogenic and inflammatory potential, due to their intrinsic biocompatibility. 2,8,[25][26][27][28] Earlier studies have demonstrated that highly ordered amyloid cross-beta structures are associated protein-specific amyloid core sequences of four to seven amino acids. [29][30][31][32] The core sequences are able to self-assemble into structurally highly similar fibrils that resemble the fibrils derived from the entire protein.…”

Marine fungi as source of new hydrophobins

“…They have been split in two groups, class I and class II, based on structural differences and properties of the aggregates they form [9]. Class I HFBs form highly insoluble aggregates that have the appearance of distinct rodlets and, similarly to amyloid fibrils, are characterized by cross β-structure [10]. These assemblies show outstanding stability and can be depolymerized in 100% trifluoroacetic acid (TFA) whereas class II HFBs form less stable polymers that are soluble in some organic solvents or SDS aqueous solution, and lack the rodlet appearance of class I HFBs [11].…”

Intrinsic disorder modulates protein self-assembly and aggregation

“…We report that flexible regions of proteins can play a major role in the avoidance of self-assembly and aggregation. In solution EAS remains monomeric but on contact with a hydrophobichydrophilic boundary, such as an air-water interface, EAS rapidly assembles into amyloid fibrils (24) that play a functional role in the fungal life cycle (23). In the organism, however, EAS selfassembly must be tightly controlled and restricted to specific conditions and appropriate locations.…”