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

Ingrid Macindoea,1, Ann H. Kwana,1, Qin Rena,b, Vanessa K. Morrisa,b, Wenrong Yangc, Joel P. Mackaya, and Margaret Sundea,b,2 aSchool of Molecular Bioscience, and bDiscipline of Pharmacology, University of Sydney, Sydney, New South Wales 2006, Australia; and cSchool of Life and Environmental Sciences, Deakin University, Geelong, Victoria 3217, Australia AUTHOR SUMMARY

Airborne fungal spores are into the amyloid form: the covered with an amphipathic sequence from residues 70–76. protein layer that confers A peptide corresponding to water resistance and this region is able to interfere facilitates dispersal and with EAS rodlet assembly, colonization (1). In the mold confirming that these residues Neurospora crassa, the fibrillar not only drive the assembly protein layer is generated by process but also directly form the self-assembly of the the stabilizing core of the hydrophobin EAS. The amyloid structure. With this hydrophobin fibrils are knowledge, we grafted the 70– termed rodlets and they have 76 sequence onto the structure an underlying protein of a non-rodlet-forming structure similar to that of the hydrophobin and we are able amyloid fibrils found in to show that this chimera disease-associated deposits protein can form rodlets. This (2). Hydrophobin rodlets are result shows that a relatively therefore functional amyloids, small segment is all that is where the structure plays an required to drive protein self- important biological role. association and fibril Understanding the self- formation. assembly of this functional In the monomer structure of amyloid will enable the EAS, the 70–76 region is engineering of biocompatible located on the edge of the hydrophobin layers with protein, hydrogen bonded to unique properties and provide the β-barrel. We postulated a strategy for inhibiting that the 70–76 region must hydrophobin assembly Fig. P1. The self-assembly of hydrophobin proteins into a monolayer undergo a conformational associated with fungal with rodlet morphology and amyloid characteristics. Hydrophobins are change in order to drive infections. In our current secreted by fungi as monomers. Upon encountering a hydrophobic: polymer assembly. To work, we have identified the hydrophilic interface, such as the surface of a spore in contact with understand the structural air, the structure of the hydrophobin monomer undergoes a change critical amyloid determinants rearrangement that within the EAS sequence and that results in the exposure of an aggregation-prone region. This accompanies rodlet formation, we have elucidated the region makes intermolecular contacts with adjacent monomers and β we determined the monomer structural changes that occur forms the amyloid -sheet core of the polymerized fibrillar structure. The remaining bulk of the protein is accommodated on the periphery structure and the dynamics of during the EAS of the rodlet. The rodlets pack together into an amphipathic an EAS mutant with impaired polymerization process. monolayer that coats the spore surface and keeps it dry during rodlet-forming ability. This We have previously dispersal in air. structure indicated that determined the structure of the monomeric form of the hydrophobin EAS (3), which conformational flexibility in demonstrated that the EAS structure is an amphipathic β- this region is associated with rodlet assembly. We have used barrel in solution (Fig. P1). Like all other class I these experimental data to provide constraints for the hydrophobins, upon association with a hydrophobic:hydrophilic interface, EAS self-assembles spontaneously into insoluble Author contributions: I.M., A.H.K., and M.S. designed research; I.M., A.H.K., Q.R., V.K.M., rodlets, which are extremely robust and can only be W.Y., and M.S. performed research; I.M., A.H.K., and M.S. analyzed data; and A.H.K., J.P.M., depolymerized by treatment with strong acids. We aimed to and M.S. wrote the paper. identify the driving force for assembly of these unique The authors declare no conflict of interest. structures and to determine the molecular structure of the This article is a PNAS Direct Submission. rodlet form. Data deposition: The lowest 20 energy structures have been deposited in the Protein We have used mutagenesis to introduce glycine residues Data Bank, www.pdb.org (PDB ID code 2LFN), and the full set of restraints in the into the EAS protein and have measured the effect of these BioMagResBank, www.bmrb.wisc.edu (accession no. 17765). mutations on the rate of hydrophobin assembly. Glycine 1I.M. and A.H.K. contributed equally to this work. residues introduce flexibility into the protein chain and are 2To whom correspondence should be addressed. E-mail: [email protected]. known to disfavor amyloid formation, allowing us to identify See full research article on page E804 of www.pnas.org. the region of EAS that drives the association of monomers Cite this Author Summary as: PNAS 10.1073/pnas.1114052109.

5152–5153 ∣ PNAS ∣ April 3, 2012 ∣ vol. 109 ∣ no. 14 www.pnas.org/cgi/doi/10.1073/pnas.1114052109 Downloaded by guest on September 24, 2021 molecular docking of multiple EAS monomers. In this way we the functional amyloid structures found in nature demonstrate PNAS PLUS have generated a model of the assembled rodlet form in which a conformational switch to the fibrillar amyloid form, the 70–76 region forms the amyloid β-sheet core of the rodlet; analogous to that seen in disease-associated the remaining bulk of the protein is accommodated on the amyloidogenesis (4). periphery and the structure is amphipathic (Fig. P1). This model is compatible with all available experimental evidence 1. Beever RE, Dempsey G (1978) Function of rodlets on the hyphae of fungal spores. Nat- regarding rodlet morphology, dimensions, and chemical ure 272:608–610. properties and demonstrates how the amyloid structure can 2. Greenwald J, Riek R (2010) Biology of amyloid: Structure, function, and regulation. arise through a simple conformational change in only one area Structure 18:1244–1260. of the protein. 3. Kwan AH, et al. (2006) Structural basis for rodlet assembly in fungal hydrophobins. Proc This work opens the door for manipulation of hydrophobin Natl Acad Sci USA 103:3621–3626. self-assembly in both biotechnology applications and 4. Liu C, Sawaya MR, Eisenberg D (2011) beta-microglobulin forms three-dimensional do- hydrophobin-mediated fungal infections. Our results show that main-swapped amyloid fibrils with disulfide linkages. Nat Struct Mol Biol 18:49–55. BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Macindoe et al. PNAS ∣ April 3, 2012 ∣ vol. 109 ∣ no. 14 ∣ 5153 Downloaded by guest on September 24, 2021