High-speed atomic force microscopy reveals surface-catalyzed elongation mechanism of fungal functional amyloid, hydrophobin RolA

Hydrophobins, a type of functional amyloid, are conserved in filamentous fungi and act as a protective coat in the fibrous form called rodlet. Rodlets form hierarchical structures where they are bundled and densely aligned, contributing to the hydrophobicity of the mycelium surface. However, the formation mechanism of the hierarchical structures is completely unknown. In this study we used high-speed atomic force microscopy to directly observe the structural dynamics of hierarchical structure formation by hydrophobin RolA from the industrial fungus Aspergillus oryzae at a single-fibril level and revealed its mechanism. The elongation of rodlets occurred at both ends and was discontinuous, alternating between periods when they could elongate (growth state) and could not elongate (pause state). This suggests an equilibrium of two distinct structural states at the rodlet ends. We also discovered an aggregation pathway, termed surface-catalyzed elongation, in which elongation is promoted by lateral interactions between bundled rodlets. Surface-catalyzed elongation decreased the energy barrier of both structural switching between growth and pause states and elongation at rodlet ends, doubling the elongation rate in bundled rodlets. The rodlet surface could be considered as a catalyst for the elongation of neighboring rodlets. Surface-catalyzed elongation could contribute to rodlet bundling, whereby rodlets tend to form oriented domain structures, and our Monte Carlo simulations confirmed this. Surface-catalyzed elongation may be a universal concept to explain the hierarchical assembly mechanism of amyloid fibrils, so it could contribute to the advancement of amyloid research in general.