The enzymatic degradation of cellulose is an important process, both ecologically and commercially. The three-dimensional structure of a cellulase, the enzymatic core of CBHII from the fungus Trichoderma reesei reveals an alpha-beta protein with a fold similar to but different from the widely occurring barrel topology first observed in triose phosphate isomerase. The active site of CBHII is located at the carboxyl-terminal end of a parallel beta barrel, in an enclosed tunnel through which the cellulose threads. Two aspartic acid residues, located in the center of the tunnel are the probable catalytic residues.

We have crystallized the ascomycete laccase from Melanocarpus albomyces with all four coppers present and determined the crystal structure at 2.4 A resolution. The enzyme is heavily glycosylated and consists of three cupredoxin-like domains, similar to those found in the Cu-depleted basidiomycete laccase from Coprinus cinereus. However, there are significant differences in the loops forming the substrate-binding pocket. In addition, the crystal structure of the M. albomyces laccase revealed elongated electron density between all three coppers in the trinuclear copper site, suggesting that an oxygen molecule binds with a novel geometry. This oxygen, required in the reaction, may enter the trinuclear site through the tunnel, which is open in the structure of the C. cinereus laccase. In contrast, the C-terminus on the M. albomyces laccase forms a plug that blocks this access.

The Ntn-hydrolases~N-terminal nucleophile! are a superfamily of diverse enzymes that has recently been characterized. All of the proteins in this family are activated autocatalytically; they contain an N-terminally located catalytic nucleophile, and they cleave an amide bond. In the present study, the structures of four enzymes of this superfamily are compared in more detail. Although the amino acid sequence homology is almost completely absent, the enzymes share a similar abba-core structure. The central b-sheets in the core were found to have different packing angles, ranging from 5 to 358. In the Ntn-hydrolases under study, eight totally conserved secondary structure units were found~region C!. Five of them were observed to contain the greatest number of conserved and functionally important residues and are therefore crucial for the structure and function of Ntn-hydrolases. Two additional regions, consisting of secondary structure units~regions A and B!, were found to be in structurally similar locations, but in different orders in the polypeptide chain. The catalytic machinery is located in the structures in a similar manner, and thus the catalytic mechanisms of all of the enzymes are probably similar. However, the substrate binding and the oxyanion hole differed partially.

Hydrophobins are small fungal proteins that are highly surface active and possess a unique ability to form amphiphilic membranes through spontaneous self-assembly. The first crystal structure of a hydrophobin, Trichoderma reesei HFBII, revealed the structural basis for the function of this amphiphilic protein-a patch consisting of hydrophobic side chains on the protein surface. Here, the crystal structures of a native and a variant T. reesei hydrophobin HFBI are presented, revealing the same overall structure and functional hydrophobic patch as in the HFBII structure. However, some structural flexibility was found in the native HFBI structure: The asymmetric unit contained four molecules, and, in two of these, an area of seven residues was displaced as compared to the two other HFBI molecules and the previously determined HFBII structure. This structural change is most probably induced by multimer formation. Both the native and the N-Cys-variant of HFBI were crystallized in the presence of detergents, but an association between the protein and a detergent was only detected in the variant structure. There, the molecules were arranged into an extraordinary detergent-associated octamer and the solvent content of the crystals was 75%. This study highlights the conservation of the fold of class II hydrophobins in spite of the low sequence identity and supports our previous suggestion that concealment of the hydrophobic surface areas of the protein is the driving force in the formation of multimers and monolayers in the self-assembly process.Keywords: hydrophobin; amphiphile; surfactant; class II; pseudomerohedral twinning; high solvent content Hydrophobins are a group of proteins with a unique property to spontaneously self-assemble into amphiphilic layers and thus invert the hydropathy of a surface. Hydrophobins are found in filamentous fungi only, and they play important roles in fungal growth, e.g., in lowering the surface tension of water to enable the growth of the hyphae into the air and the coating of the surfaces of aerial hyphae to conceal the hydrophilic cell wall in the air environment (Wösten et al. 1999;Linder et al. 2005). A fungal species may carry several hydrophobin genes, expressed at different times during growth, located in different parts of fungi, and targeted for a specific function. Hydrophobins are nontoxic but may act in pathogenic infections by mediating the attachment to the host organism (Ebbole 1997).The unique properties of hydrophobins make them potential candidates for various medical and technical Reprint requests to: Juha Rouvinen, Department of Chemistry, University of Joensuu, P.O. Box 111, 80101 Joensuu, Finland; e-mail: juha.rouvinen@joensuu.fi; fax: +358-13-251-3390.Abbreviations: LDAO, lauryldimethylamine oxide; OSG, 1-S-octylb-D-thioglucoside; PDB, the Protein Data Bank; RMSD, root-meansquare distance; RP-HPLC, reversed-phase high-performance liquid chromatography; SAA, solvent accessible area.Article published online ahead of print. Article and publication date are a...

Hydrophobins are proteins specific to filamentous fungi. Hydrophobins have several important roles in fungal physiology, for example, adhesion, formation of protective surface coatings, and the reduction of the surface tension of water, which allows growth of aerial structures. Hydrophobins show remarkable biophysical properties, for example, they are the most powerful surface-active proteins known. To this point the molecular basis of the function of this group of proteins has been largely unknown. We have now determined the crystal structure of the hydrophobin HFBII from Trichoderma reesei at 1.0 Å resolution. HFBII has a novel, compact single domain structure containing one ␣-helix and four antiparallel ␤-strands that completely envelop two disulfide bridges. The protein surface is mainly hydrophilic, but two ␤-hairpin loops contain several conserved aliphatic side chains that form a flat hydrophobic patch that makes the molecule amphiphilic. The amphiphilicity of the HFBII molecule is expected to be a source for surface activity, and we suggest that the behavior of this surfactant is greatly enhanced by the selfassembly that is favored by the combination of size and rigidity. This mechanism of function is supported by atomic force micrographs that show highly ordered arrays of HFBII at the air water interface. The data presented show that much of the current views on structure function relations in hydrophobins must be re-evaluated.