Ossi Turunen

The ezrin protein family: membrane-cytoskeleton interactions and disease associations

Ezrin, radixin, moesin and merlin form a subfamily of conserved proteins in the band 4.1 superfamily. Ezrin protein subfamily members act as linkers between the plasma membrane and the cytoskeleton. Members of the subfamily have been shown to interact with each other, with cell adhesion molecules such as CD44 and with F-actin. Recent data indicate that intercellular adhesion molecules 1 and 2 also interact with ezrin. The proteins are also involved in the redistribution of intercellular adhesion molecules and the organization of cell membrane structures. Merlin is a tumor suppressor that is involved in tumorigenesis of schwannomas and meningiomas. Merlin has the same overall protein structure as the other proteins in the subfamily but may have partially distinct functions.

A combination of weakly stabilizing mutations with a disulfide bridge in the α-helix region of Trichoderma reesei endo-1,4-β-xylanase II increases the thermal stability through synergism

Thermal stability and other functional properties of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII; family 11) were studied by designed mutations. Mutations at three positions were introduced to the XYNII mutant containing a disulfide bridge (S110C-N154C) in the alpha-helix. The disulfide bridge increased the half-life of XYNII from less than 1 min to 14 min at 65 degrees C. An additional mutation at the C-terminus of the alpha-helix (Q162H or Q162Y) increased the half-life to 63 min. Mutations Q162H and Q162Y alone had a stabilizing effect at 55 degrees C but not at 65 degrees C. The mutations N11D and N38E increased the half-life to about 100 min. Due to the stabilizing mutations the pH stability increased in a wide pH range, but at the same time the activity decreased both in acidic and neutral-alkaline pH, the pH optimum being at pH region 5-6. There was no essential difference between the specific activities of the mutants and the wild-type XYNII.

Improved thermal performance of Thermomyces lanuginosus GH11 xylanase by engineering of an N-terminal disulfide bridge

In order to increase the stability of thermophilic Thermomyces lanuginosus GH11 xylanase, TLX, a disulfide bridge Q1C-Q24C was introduced into the N-terminal region of the enzyme. The apparent temperature optimum shifted upwards at pH 6.5 by about 10°C to 75°C. The resistance to thermal inactivation also increased by about 10°C. The melting temperature measured by CD spectroscopy increased from 66 to 74°C. Therefore the N-terminal disulfide bridge increased both kinetic and thermodynamic stability almost equally. At pH 8 and 70°C, the disulfide bridge increased the enzyme half-life 20-fold in the presence of substrate. In contrast to the situation in acidic-neutral pH, the substrate decreased the thermostability of xylanases in alkaline pH. The upper limit for the performance of the disulfide bridge mutant at pH 9 was 75°C. This study showed that N-terminal disulfide bridges can stabilize even thermostable family GH11 xylanases.

Influence of pH on the production of xylanases by Trichoderma reesei Rut C-30

Trichoderma reesei Rut C-30 was cultivated in bioreactors at different pH on a medium with lactose as the main carbon source. Compared to an earlier study, in which T. reesei Rut C-30 was cultivated using polysaccharides (cellulose or xylan) as the main carbon sources, we now report a slightly lower pH value for maximal xylanase levels. The highest xylanase activity (IU/ml) on the lactose-based medium was observed at pH 6.0 compared to pH 7.0 on the polysaccharide-based media. When the pattern of different xylanases was analyzed by isoelectric focusing and activity zymogram, we observed that a low pH (4.0) favoured the production of xylanase I, whilst a high pH (6.0) favoured the production of xylanase III. Xylanase II was clearly produced at both pH values. The results at pH 4 and 6 correlate with the pH activity profiles of xylanase I, II and III. Hence, the different T. reesei xylanases were produced according to which enzyme is most active in that particular environment.

Structure-function relationships in the ezrin family and the effect of tumor-associated point mutations in neurofibromatosis 2 protein

Ezrin, radixin and moesin (ERM proteins) link cell adhesion molecules to the cytoskeleton, modulate cell morphology and cell growth and are involved in Rho-mediated signal transduction. Merlin, the tumor suppressor in neurofibromatosis 2, is a diverged member of the ezrin family, but its function is at least partially similar to the ERM proteins. In the N-domain, the ezrin family belongs to the band 4.1 superfamily. Secondary structure predictions made separately for the ezrin and band 4.1-tyrosine phosphatase families give a similar pattern for the homologous N-domains, indicating that both families have a similar binding site for the integral membrane proteins. The alpha-domain shows a strong coiled-coil prediction, that can be involved in the protein dimerization. The C-terminal actin-binding site in the ERM proteins and the actin-binding helix in the villin headpiece have a common amino acid motif. In merlin, the published tumor-associated single amino acid mutations in the N-domain are located in the conserved sites, and they affect mainly the predicted helices and strands, indicating that these mutations cause the disease primarily by disturbing the protein structure. In the alpha- and C-domains, some of the mutations break the helical structures. Some known mutations are observed at a site potentially interacting with cell adhesion molecules. We will also discuss the implications of the evolutionary information and the actin-binding models in the ezrin family.

Protein engineering: opportunities and challenges

The extraordinary properties of natural proteins demonstrate that life-like protein engineering is both achievable and valuable. Rapid progress and impressive results have been made towards this goal using rational design and random techniques or a combination of both. However, we still do not have a general theory on how to specify a structure that is suited to a target function nor can we specify a sequence that folds to a target structure. There is also overreliance on the Darwinian blind search to obtain practical results. In the long run, random methods cannot replace insight in constructing life-like proteins. For the near future, however, in enzyme development, we need to rely on a combination of both.

Thermostability Improvement of a Streptomyces Xylanase by Introducing Proline and Glutamic Acid Residues

ABSTRACT Protein engineering is commonly used to improve the robustness of enzymes for activity and stability at high temperatures. In this study, we identified four residues expected to affect the thermostability of Streptomyces sp. strain S9 xylanase XynAS9 through multiple-sequence analysis (MSA) and molecular dynamic simulations (MDS). Site-directed mutagenesis was employed to construct five mutants by replacing these residues with proline or glutamic acid (V81P, G82E, V81P/G82E, D185P/S186E, and V81P/G82E/D185P/S186E), and the mutant and wild-type enzymes were expressed in Pichia pastoris. Compared to the wild-type XynAS9, all five mutant enzymes showed improved thermal properties. The activity and stability assays, including circular dichroism and differential scanning calorimetry, showed that the mutations at positions 81 and 82 increased the thermal performance more than the mutations at positions 185 and 186. The mutants with combined substitutions (V81P/G82E and V81P/G82E/D185P/S186E) showed the most pronounced shifts in temperature optima, about 17°C upward, and their half-lives for thermal inactivation at 70°C and melting temperatures were increased by >9 times and approximately 7.0°C, respectively. The mutation combination of V81P and G82E in adjacent positions more than doubled the effect of single mutations. Both mutation regions were at the end of long secondary-structure elements and probably rigidified the local structure. MDS indicated that a long loop region after positions 81 and 82 located at the end of the inner β-barrel was prone to unfold. The rigidified main chain and filling of a groove by the mutations on the bottom of the active site canyon may stabilize the mutants and thus improve their thermostability.

The crystal structure of acidic β-galactosidase from Aspergillus oryzae.

The crystal structure of the industrially important Aspergillus oryzae β-galactosidase has been determined at 2.60 Å resolution. The Ao-β-gal is a large (985 residues) monomeric multi-domain enzyme that has a catalytic (α/β)8-barrel domain. An electron density map revealed extensive N-glycosylation between the domain interfaces suggesting that the oligosaccharide-chains would have a stabilizing role for the structure of Ao-β-gal. Comparison of structure with other β-galactosidase structures of glycoside hydrolase family 35 revealed a number of hydrophobic residues, which may contribute favorably to the stabilization of the structure. The role of a high number of acidic residues in Ao-β-gal is also discussed.

Thermostabilization of extremophilic Dictyoglomus thermophilum GH11 xylanase by an N-terminal disulfide bridge and the effect of ionic liquid [emim][OAc on the enzymatic performance

In the present study, an extremophilic GH11 xylanase was stabilized by an engineered N-terminal disulphide bridge. The effect of the stabilization was then tested against high temperatures and in the presence of a biomass-dissolving ionic liquid, 1-ethyl-3-methylimidazolium acetate ([emim]OAc). The N-terminal disulfide bridge increased the half-life of a GH11 xylanase (XYNB) from the hyperthermophilic bacterium Dictyoglomus thermophilum by 10-fold at 100°C. The apparent temperature optimum increased only by ∼5°C, which is less than the corresponding increase in mesophilic (∼15°C) and moderately thermophilic (∼10°C) xylanases. The performance of the enzyme was increased significantly at 100-110°C. The increasing concentration of [emim]OAc almost linearly increased the inactivation level of the enzyme activity and 25% [emim]OAc inactivated the enzyme almost fully. On the contrary, the apparent temperature optimum did not decrease to a similar extent, and the degree of denaturation of the enzyme was also much lower according to the residual activity assays. Also, 5% [emim]OAc largely counteracted the benefit obtained by the stabilizing disulfide bridge in the temperature-dependent activity assays, but not in the stability assays. Km was increased in the presence of [emim]OAc, indicating that [emim]OAc interfered the substrate-enzyme interactions. These results indicate that the effect of [emim]OAc is targeted more to the functioning of the enzyme than the basic stability of the hyperthermophilic GH11 xylanase.

Crystal structures of Trichoderma reesei β-galactosidase reveal conformational changes in the active site

We have determined the crystal structure of Trichoderma reesei (Hypocrea jecorina) β-galactosidase (Tr-β-gal) at a 1.2Å resolution and its complex structures with galactose, IPTG and PETG at 1.5, 1.75 and 1.4Å resolutions, respectively. Tr-β-gal is a potential enzyme for lactose hydrolysis in the dairy industry and belongs to family 35 of the glycoside hydrolases (GH-35). The high resolution crystal structures of this six-domain enzyme revealed interesting features about the structure of Tr-β-gal. We discovered conformational changes in the two loop regions in the active site, implicating a conformational selection-mechanism for the enzyme. In addition, the Glu200, an acid/base catalyst showed two different conformations which undoubtedly affect the pK(a) value of this residue and the catalytic mechanism. The electron density showed extensive glycosylation, suggesting a structure stabilizing role for glycans. The longest glycan showed an electron density that extends to the eighth monosaccharide unit in the extended chain. The Tr-β-gal structure also showed a well-ordered structure for a unique octaserine motif on the surface loop of the fifth domain.