Crystal Structures of Hydrophobin HFBII in the Presence of Detergent Implicate the Formation of Fibrils and Monolayer Films

Abstract

Hydrophobins are small, amphiphilic proteins secreted by filamentous fungi. Their functionality arises from a patch of hydrophobic residues on the protein surface. Spontaneous selfassembly of hydrophobins leads to the formation of an amphiphilic layer that remarkably reduces the surface tension of water. We have determined by x-ray diffraction two new crystal structures of Trichoderma reesei hydrophobin HFBII in the presence of a detergent. The monoclinic crystal structure (2.2 Å resolution, R ‫؍‬ 22, R free ‫؍‬ 28) is composed of layers of hydrophobin molecules where the hydrophobic surface areas of the molecules are aligned within the layer. Viewed perpendicular to the aligned hydrophobic surface areas, the molecules in the layer pack together to form six-membered rings, thus leaving small pores in the layer. Similar packing has been observed in the atomic force microscopy images of the self-assembled layers of class II hydrophobin, indicating that the crystal structure resembles that of natural hydrophobin film. The orthorhombic crystal structure (1.0 Å resolution, R ‫؍‬ 13, R free ‫؍‬ 15) is composed of fiber-like arrays of protein molecules. Rodlet structures have been observed on amphiphilic layers formed by class I hydrophobins; fibrils of class II hydrophobins appear by vigorous shaking. We propose that the structure of the fibrils and/or rodlets is similar to that observed in the crystal structure.Filamentous fungi secrete small proteins, hydrophobins, that have a unique ability to spontaneously form amphiphilic layers on hydrophobic-hydrophilic interfaces (1). Hydrophobins are distributed in two classes according to the pattern of hydrophobic and hydrophilic amino acid residues in the protein sequence and the dissociation resistance of the assembled amphiphilic layers (2). The pioneering work (3) with the class I hydrophobin SC3 from Schizophyllum commune revealed the dual role served by this protein in the growth of the fungal hypha: hydrophobins reduce the surface tension of water and provide a protective coating on the hyphal surface, once exposed to air.Hydrophobins are an attractive target of study due to their vast application potential provided by their unique characteristics (4). Examples include anti-fouling applications, coatings that increase biocompatibility in medical instruments, coatings in drug delivery, surface patterning, use in products of personal care as emulsifiers, use in immobilization techniques, and as very potent foam-forming agents (5, 6). In addition, protein purification of hydrophobin fusion proteins in aqueous twophase systems has been demonstrated (7).So that we understand the mechanism of the function of hydrophobins, the structural information of these proteins is crucial. The first hydrophobin structure was determined by x-ray diffraction in 2004 (PDB codes 1R2M, 2B97) (8, 9), describing a class II hydrophobin HFBII from Trichoderma reesei. Now, the structure of a class I hydrophobin EAS from Neurospora crassa has been determined by the NMR method (PDB code 2FMC) (10...

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

“…Therefore, it is likely that a conformational change involving a larger proportion of the Cys7-Cys8 loop can take place upon contact with a suitable interface. In the crystal structures of the class II hydrophobins HFBI and HFBII solved in the presence of detergent, the Cys7-Cys8 loop in some of the monomers is observed in an extended conformation (35,36). While these class II hydrophobins do not form rodlets and the sequence of this corresponding region in the class II hydrophobins is not predicted to be amyloidogenic, the observation does indicate that this region of the protein is inherently mobile in response to a hydrophobic environment.…”

Structure-Function Relationships in Hydrophobins: Probing the Role of Charged Side Chains

“…Oligomerization was chosen as a parameter for understanding hydrophobins because structural (12) and functional (18) data show that solution interactions are characteristic properties, but the exact mechanisms are not completely understood. The AF4 method is a good alternative to size exclusion chromatography analysis, as the absence of a matrix minimizes shear forces, allowing the observation of weak interactions, and also minimizes interaction of the sample with the stationary phase.…”

“…Structural analyses of hydrophobins (11)(12)(13)(14) have given significant insight into how hydrophobins function as surface-active and surface-adhering proteins and what confers their special properties. They have a clearly distinguishable hydrophobic patch on the surface that suggests how the hydrophobin can act as an amphiphilic particle-like structure.…”

Fungal and mushroom hydrophobins: A review

“…The hydrophobic part including two β-hairpins between cysteine (3-4) and (7-8) is observed in tertiary structure of both class I and II hydrophobins. And a hydrophilic part which includes one α-helix between cysteine (4-5) is present in class II hydrophobins only but not class I hydrophobins (Kallio et al, 2007;Linder, 2009).…”

“…The most intrinsic property of hydrophobins is the self-assembled amphipathic membranes at hydrophobic-hydrophilic surfaces and interfaces (Linder, 2009). These tough, ordered and robust membranes are crystalline and viscoelastic which are important for aerial growth of fungi (Kallio et al, 2007). Surface membranes on the air-water interface are monolayers for class II hydrophobins but mono/ multilayers for class I (Garbe et al, 2009).…”