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

Markus B. Linder,Juha Rouvinen,Johanna M. Kallio

doi:10.1074/jbc.m704238200

Abstract

Hydrophobins are small, amphiphilic proteins secreted by filamentous fungi. Their functionality arises from a patch of hydrophobic residues on the protein surface. Spontaneous self-assembly 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 (2.2A 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 A 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.

Highlights

Hydrophobins are an attractive target of study due to their vast application potential provided by their unique characteristics [4]
Based on the x-ray structure of HFBII and the x-ray structures of native and variant forms of HFBI (PDB codes 2FZ6, 2GVM) [11], which is a class II hydrophobin from T. reesei, we have previously proposed that oligomerization in solution is an important property of hydrophobins
The Monoclinic Crystal Form of HFBII—The structure of the monoclinic crystal form has been deposited with the PDB under code 2PL6

Summary

Introduction

Hydrophobins are an attractive target of study due to their vast application potential provided by their unique characteristics [4]. Class I and II hydrophobins, on the basis of determined structures, seem to share a common fold with a small ␤-barrel forming the core structure and being reinforced by four disulfide bridges formed by the eight conserved cysteine residues that are characteristic of all hydrophobin sequences. The hydrophobic patch covers ϳ18% of the total surface area, and contributing residues are mainly located between the third and the fourth and the seventh and the eighth cysteines in the protein sequence (supplemental data). This is the area in which the disordered loops occur in the class I EAS structure (Met-22-Ser-42 and Val-65-Phe-72), and it is difficult to interpret whether the functional site is the same in both classes. The sequence comparison reveals that the segment between the fourth and the fifth cysteines in class I hydropho-

Methods

Results

Conclusion