The effect of changing the hydrophobic S1' subsite of thermolysin-like proteases on substrate specificity.

The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.

Substrate specificity in the highly heterogeneous M4 peptidase family is determined by a small subset of amino acids.

The members of the M4 peptidase family are involved in processes as diverse as pathogenicity and industrial applications. For the first time a number of M4 family members, also known as thermolysin-like proteases, has been characterized with an identical substrate set and a uniform set of assay conditions. Characterization with peptide substrates as well as high performance liquid chromatography analysis of beta-casein digests shows that the M4 family is a homogeneous family in terms of catalysis, even though there is a significant degree of amino acid sequence variation. The results of this study show that differences in substrate specificity within the M4 family do not correlate with overall sequence differences but depend on a small number of identifiable amino acids. Indeed, molecular modeling followed by site-directed mutagenesis of one of the substrate binding pocket residues of the thermolysin-like proteases of Bacillus stearothermophilus converted the catalytic characteristics of this variant into that of thermolysin.

Characteristics of the biologically active 35-kDa metalloprotease virulence factor from Listeria monocytogenes.

Listeria monocytogenes, a facultative intracellular pathogen, synthesizes an extracellular protease which is responsible for the maturation of phosphatidylcholine phospholipase C (lecithinase), a virulence factor involved in cell-to-cell spread. This work describes the environmental parameters necessary for increased production of mature, 35-kDa active protease in strains of L. monocytogenes, and its detection using polyclonal antibodies raised against Bacillus subtilis neutral protease. High performance liquid affinity chromatography was exploited to isolate the biologically active form of the mature protease, which was then subjected to biochemical characterization using casein as a substrate. The protease is a zinc-dependent metalloprotease which degrades casein over a wide range of temperatures and pH values. It can also degrade actin, the most abundant protein in many eukaryotic cells. The Listeria protease was shown to exhibit a high thermal stability and a relatively narrow substrate specificity. A three-dimensional model built on the basis of the homology with thermolysin was used to understand the structural basis of these characteristics.

Two allelic forms of the aureolysin gene (aur) within Staphylococcus aureus.

Proteinases of Staphylococcus aureus are emerging as potential virulence factors which may be involved in the pathogenecity of staphylococcal diseases. We describe here the structure of the gene encoding the metalloproteinase referred to as aureolysin. This gene occurs in two allelic forms and is strongly conserved among S. aureus strains, implying the possibility that the proteinase may have important housekeeping functions.

Characterization of a novel stable biocatalyst obtained by protein engineering.

Protein engineering is a powerful tool for the improvement of the properties of biocatalysts. Previously we have applied protein engineering technologies to obtain an extremely stable variant of the thermolysin-like protease from Bacillus stearothermophilus [Van den Burg, Vriend, Veltman, Venema and Eijsink (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2056-2060]. This variant is much more resistant to denaturing conditions (temperature and denaturing agents) than the wild-type enzyme. An extensive enzymic characterization was undertaken to explore the suitability of the variant as a biocatalyst at high temperatures. By comparing a range of variants with increasing thermal stabilities we show that the additivity of the mutations is accompanied by an increase in activity at elevated temperatures in accordance with the Arrhenius law. The results suggest that the constructed protease variants could be suitable alternatives to proteases that are currently used industrially. Our studies demonstrate how protein engineering can be exploited to obtain high-performance biocatalysts.

Probing the unfolding region in a thermolysin-like protease by site-specific immobilization.

Protein stabilization by immobilization has been proposed to be most effective if the protein is attached to the carrier at that region where unfolding is initiated. To probe this hypothesis, we have studied the effects of site-specific immobilization on the thermal stability of mutants of the thermolysin-like protease from Bacillus stearothermophilus (TLP-ste). This enzyme was chosen because previous studies had revealed which parts of the molecule are likely to be involved in the early steps of thermal unfolding. Cysteine residues were introduced by site-directed mutagenesis into various positions of a cysteine-free variant of TLP-ste. The mutant enzymes were immobilized in a site-specific manner onto Activated Thiol-Sepharose. Two mutants (T56C, S65C) having their cysteine in the proposed unfolding region of TLP-ste showed a 9- and 12-fold increase in half-lives at 75 degrees C due to immobilization. The stabilization by immobilization was even larger (33-fold) for the T56C/S65C double mutant enzyme. In contrast, mutants containing cysteines in other parts of the TLP-ste molecule (N181C, S218C, T299C) showed only small increases in half-lives due to immobilization (maximum 2.5-fold). Thus, the stabilization obtained by immobilization was strongly dependent on the site of attachment. It was largest when TLP-ste was fixed to the carrier through its postulated unfolding region. The concept of the unfolding region may be of general use for the design of strategies to stabilize proteins.

Early steps in the unfolding of thermolysin-like proteases.

Several series of site-directed mutations in thermolysin-like proteases are presented that show remarkable nonadditivity in their effect on thermal stability. A simple model is proposed that relates this nonadditivity to the occurrence of independent partial unfolding processes that occur in parallel at elevated temperatures. To prove this model, a thermolysin-like protease was designed in which two mutations located approximately 35 A apart in the structure individually exert small stabilizing effects of 2.3 and 4. 1 degreesC, respectively, but when combined stabilize the protease by 14.6 degreesC. This overadditivity, which follows directly from the model, confirms that unfolding of this engineered protease starts in parallel at two different regions of the protein.

Rendering one autolysis site in Bacillus subtilis neutral protease resistant to cleavage reveals a new fission.

Autolytic degradation of the thermolysin-like proteinase of Bacillus subtilis (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously we have reported five cleavage sites in TLP-sub [Van den Burg et al. (1990) Biochem. J. 272, 93-97]. In an attempt to render the enzyme less susceptible to autolytic breakdown, one of the fission sites, located between Leu-156 and Ile-157, was modified by replacing Ile-157, C-terminally located with respect to the fission site, by an Asp residue. Aspartic acid is less preferred at this position with respect to the substrate preference of TLP-sub. Modelling studies indicated that this mutation was unlikely to cause conformational changes in the enzyme. Although the 156-157 fission was not observed in the mutant enzyme, a new fission site, between Gly-148 and Val-149, was now observed.

Probing catalytic hinge bending motions in thermolysin-like proteases by glycine --> alanine mutations.

The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in "closure" and "opening" of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly --> Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-L-glycyl-L-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.

A single calcium binding site is crucial for the calcium-dependent thermal stability of thermolysin-like proteases.

Thermostable thermolysin-like proteases (TLPs), such as the TLP of Bacillus stearothermophilus CU-21 (TLP-ste), bind calcium in one double (Ca1,2) and two single (Ca3, Ca4) calcium binding sites. The single sites are absent in thermolabile TLPs, suggesting that they are determinants of (variation in) TLP stability. Mutations in the Ca3 and Ca4 sites of TLP-ste indeed reduced thermal stability, but only mutations in the Ca3 site affected the calcium-dependence of stability. The predominant effect of the Ca3 site results from the fact that the Ca3 site is part of a region of TLP-ste, which unfolding is crucial for thermal inactivation. Thermal inactivation is not caused by the absence of calcium from the Ca3 site per se, but rather by unfolding of a region of TLP-ste for which stability depends on the occupancy of the Ca3 site. In accordance with this concept is the observation that the effects of mutations in the Ca3 site could be compensated by stabilizing mutations near this site. In addition, it was observed that the contribution of calcium binding to the Ca3 was substantially reduced in extremely stable TLP-ste variants containing multiple stabilizing mutations in the Ca3 region. Apparently, in these latter variants, unfolding of the Ca3 region contributes little to the overall process of thermal inactivation.

Engineering an enzyme to resist boiling.

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

Mutational analysis of a surface area that is critical for the thermal stability of thermolysin-like proteases.

Site-directed mutagenesis was used to assess the contribution of individual residues and a bound calcium in the 55-69 region of the thermolysin-like protease of Bacillus stearothermophilus (TLP-ste) to thermal stability. The importance of the 55-69 region was reflected by finding that almost all mutations had drastic effects on stability. These effects (both stabilizing and destabilizing) were obtained by mutations affecting main chain flexibility, as well as by mutations affecting the interaction between the 55-69 region and the rest of the protease molecule. The calcium-dependency of stability could be largely abolished by mutating one of its ligands (Asp57 or Asp59). In the case of the Asp57-->Ser mutation, the accompanying loss in stability was modest compared with the effects of other destabilizing mutations or the effects of (combinations of) stabilizing mutations. The detailed knowledge of the stability-determining region of TLP-ste permits effective rational design of stabilizing mutations, which, presumably, are also useful for related TLP such as thermolysin. This is demonstrated by the successful design of a stabilizing salt bridge involving residues 65 and 11.

Design of thermolabile bacteriophage repressor mutants by comparative molecular modeling.

Comparative molecular modeling was performed with repressor protein Rro of the temperate Lactococcus lactis bacteriophage r1t using the known 3D-structures of related repressors in order to obtain thermolabile derivatives of Rro. Rro residues presumed to stabilize a nonhomologous but structurally conserved hydrophobic pocket, which was shown to be important for thermostability of the Escherichia coli bacteriophage lambda repressor CI, were randomized. Of the derivatives that exhibited various temperature-sensitive phenotypes, one was shown to hold promise for both fundamental and industrial applications that require the controlled production of (heterologous) proteins in L. lactis.

Extreme stabilization of a thermolysin-like protease by an engineered disulfide bond.

The thermal inactivation of broad specificity proteases such as thermolysin and subtilisin is initiated by partial unfolding processes that render the enzyme susceptible to autolysis. Previous studies have revealed that a surface-located region in the N-terminal domain of the thermolysin-like protease produced by Bacillus stearothermophilus is crucial for thermal stability. In this region a disulfide bridge between residues 8 and 60 was designed by molecular modelling, and the corresponding single and double cysteine mutants were constructed. The disulfide bridge was spontaneously formed in vivo and resulted in a drastic stabilization of the enzyme. This stabilization presents one of the very few examples of successful stabilization of a broad specificity protease by a designed disulfide bond. We propose that the success of the present stabilization strategy is the result of the localization and mutation of an area of the molecule involved in the partial unfolding processes that determine thermal stability.

Engineering thermolysin-like proteases whose stability is largely independent of calcium.

Thermal stability of the thermolysin-like protease produced by Bacillus stearothermophilus (TLP-ste) is highly dependent on calcium at concentrations in the millimolar range. We describe the rational design and production of a fully active TLP-ste variant whose stability is only slightly dependent on calcium concentration.

Analysis of structural determinants of the stability of thermolysin-like proteases by molecular modelling and site-directed mutagenesis.

The thermolysin-like protease (TLP) produced by Bacillus stearothermophilus CU21 (TLP-ste) differs at 43 positions from the more thermally stable thermolysin (containing 316 residues in total). Of these differences, 26 were analysed by studying the effect of replacing residues in TLP-ste by the corresponding residues in thermolysin. Several stabilizing mutations were identified but, remarkably, considerable destabilizing mutational effects were also found. A Tyr-rich three residue insertion in TLP-ste (the only deletional/insertional difference between the two enzymes) appeared to make an important contribution to the stability of the enzyme. Mutations with large effects on stability were all localized in the beta-pleated N-terminal domain of TLP-ste, confirming observations that this domain has a lower intrinsic stability than the largely alpha-helical C-terminal domain. Rigidifying mutations such as Gly58-->Ala and Ala69-->Pro were among the most stabilizing ones. Apart from this observation, the analyses did not reveal general rules for stabilizing proteins. Instead, the results highlight the importance of context in evaluating the stability effects of mutations.

Protein stabilization by hydrophobic interactions at the surface.

The contribution of the solvent-exposed residue 63 to thermal stability of the thermolysin-like neutral protease of Bacillus stearothermophilus was studied by analyzing the effect of twelve different amino acid substitutions at this position. The thermal stability of the enzyme was increased considerably by introducing Arg, Lys or bulky hydrophobic amino acids. In general, the effects of the mutations showed that hydrophobic contacts in this surface-located region of the protein are a major determinant of thermal stability. This observation contrasts with general concepts concerning the contribution of surface-located residues and surface hydrophobicity to protein stability and indicates new ways for protein stabilization by site-directed mutagenesis.

Introduction of disulfide bonds into Bacillus subtilis neutral protease.

The effects of engineered disulfide bonds on autodigestion and thermostability of Bacillus subtilis neutral protease (NP-sub) were studied using site-directed mutagenesis. After modelling studies two locations that might be capable of forming disulfide bonds, both near previously determined autodigestion sites in NP-sub, were selected for the introduction of cysteines. Analysis of mutant enzymes showed that disulfide bonds were indeed formed in vivo, and that the mutant enzymes were fully active. The introduced disulfides did not alter the autodigestion pattern of the NP-sub. All mutant NP-subs exhibited decreased thermostability, which, by using reducing agents, was shown to be caused by the introduction of the cysteines and not by the formation of the disulfides. Mutants containing one cysteine exhibited intermolecular disulfide formation at elevated temperatures, which, however, was shown not to be the cause of the decreased thermostability. Combining the present data with literature data, it would seem that the introduction of disulfide bridges is unsuitable for the stabilization of proteases. Possible explanations for this phenomenon are discussed.

Effects of changing the interaction between subdomains on the thermostability of Bacillus neutral proteases.

Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to alter the interaction between the middle and C-terminal subdomain of these enzymes. In all Nprs a cluster of hydrophobic contacts centered around residue 315 contributes to this interaction. In thermostable Nprs (like Npr-ste) a 10 residue beta-hairpin, covering the domain interface, makes an additional contribution. The hydrophobic residue at position 315 was replaced by smaller amino acids. In addition, the beta-hairpin was deleted from Npr-ste and inserted into Npr-sub. The changes in thermostability observed after these mutations confirmed the importance of the hydrophobic cluster and of the beta-hairpin for the structural integrity of Nprs. Combined mutants showed that the effects of individual mutations affecting the interaction between the subdomains were not additive. The effects on thermostability decreased as the strength of the subdomain interaction increased. The results show that once the subdomain interface is sufficiently stabilized, additional stabilizing mutations at the same interface do not further increase thermostability. The results are interpreted on the basis of a model for the thermal inactivation of neutral proteases, in which it is assumed that inactivation results from the occurrence of local unfolding processes that render these enzymes susceptible to autolysis.

The effect of cavity-filling mutations on the thermostability of Bacillus stearothermophilus neutral protease.

Cavities in the hydrophobic core of the neutral protease of Bacillus stearothermophilus were analyzed using a three-dimensional model that was inferred from the crystal structure of thermolysin, the highly homologous neutral protease of B. thermoproteolyticus (85% sequence identity). Site-directed mutagenesis was used to fill some of these cavities, thereby improving hydrophobic packing in the protein interior. The mutations had small effects on the thermostability, even after drastic changes, such as Leu284----Trp and Met168----Trp. The effects on T50, the temperature at which 50% of the enzyme is irreversibly inactivated in 30 min, ranged from 0.0 to +0.4 degrees C. These results can be explained by assuming that the mutations have positive and negative structural effects of approximately the same magnitude. Alternatively, it could be envisaged that the local unfolding steps, which render the enzyme susceptible towards autolysis and which are rate limiting in the process of thermal inactivation, are only slightly affected by alterations in the hydrophobic core.