Structural basis for rodlet assembly in fungal hydrophobins

Abstract

Class I hydrophobins are a unique family of fungal proteins that form a polymeric, water-repellent monolayer on the surface of structures such as spores and fruiting bodies. Similar monolayers are being discovered on an increasing range of important microorganisms. Hydrophobin monolayers are amphipathic and particularly robust, and they reverse the wettability of the surface on which they are formed. There are also significant similarities between these polymers and amyloid-like fibrils. However, structural information on these proteins and the rodlets they form has been elusive. Here, we describe the three-dimensional structure of the monomeric form of the class I hydrophobin EAS. EAS forms a beta-barrel structure punctuated by several disordered regions and displays a complete segregation of charged and hydrophobic residues on its surface. This structure is consistent with its ability to form an amphipathic polymer. By using this structure, together with data from mutagenesis and previous biophysical studies, we have been able to propose a model for the polymeric rodlet structure adopted by these proteins. X-ray fiber diffraction data from EAS rodlets are consistent with our model. Our data provide molecular insight into the nature of hydrophobin rodlet films and extend our understanding of the fibrillar beta-structures that continue to be discovered in the protein world.

Fig. 1.

Solution structure of EAS. (A) Sequences of EAS and the class II hydrophobin HFBII, indicating the conserved disulfide bonding pattern. (B) Overlay of the 20 lowest-energy conformers of EAS. (C) Ribbon diagram of EAS. Cys side chains are shown as orange sticks. Positively and negatively charged residues are shown as blue and red sticks, respectively. (D) Electrostatic surface of EAS. The clustering of charged residues is apparent. (E) Overlay of EAS with the x-ray crystal structure of HFBII. The minimized average structure of EAS is shown in cyan, and HFBII (Protein Data Bank ID code 1R2M) is shown in yellow. Cys residues are shown in neon.

Fig. 2.

Possible model for EAS rodlet formation. Schematic representation of EAS monomers stacking at an air:water interface. Red circles indicate charged residues. The two disordered loops might “add on” to the barrel, forming additional β-structure that H-bonds to the next monomer. Adjacent monomers are colored blue and red for clarity.

Fig. 3.

Molecular structures of EAS rodlet models. Structures of EAS dimers were calculated by using the monomer NMR restraint list supplemented with putative H-bonds. (A) Five lowest-energy structures and ribbon diagram of the dimeric model without incorporation of the flexible loops in the repeating β-structure. (D) As in A, but with incorporation of the flexible loops in the repeating β-structure in the dimeric model. (B and E) Ribbon diagram of the models in the same orientation as A and D, respectively. (C and F) Electrostatic potential diagram of the dimeric models, in the same orientation as A and D, respectively.

Fig. 4.

X-ray fiber diffraction of EAS rodlets. (Left) Pattern obtained from pelleted, preformed rodlets. (Right) Pattern obtained from rodlets prepared in a capillary in a magnetic field. Both patterns have a strong reflection at 4.7 Å, from the spacing between adjacent β-strands in a β-sheet. A very weak and diffuse inner reflection is seen at ≈10 Å (left), probably arising from an intersheet spacing. The 27-Å reflection is consistent with the dimensions of hydrophobin monomers arranged along the long axis of the fibril.