Engineering high performance variants of Bacillusthermolysin-like proteases Veltman, Oene Robert

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1 University of Groningen Engineering high performance variants of Bacillusthermolysin-like proteases Veltman, Oene Robert IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 1997 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Veltman, O. R. (1997). Engineering high performance variants of Bacillusthermolysin-like proteases. Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Construction of extremely stable TLP-ste variants Chapter 4 Structural Determinants Of The Stability Of Thermolysin-Like Proteinases Published in: Nature Structural Biology (1995) 2,

3 Chapter 4 SUMMARY Thermolysin is a member of a family of homologous proteinases which differ in their resistance to thermally induced unfolding and subsequent autolytic degradation. Sitedirected mutagenesis studies of the thermolysin-like proteinase (TLP) from Bacillus stearothermophilus (TLP-ste) show that its reduced resistance to thermally induced autolysis, as compared to thermolysin, is due to only some of the naturally occurring 44 amino-acid differences between them. In fact TLP-ste becomes more resistant than thermolysin by mutation of just a few of these amino-acids. The crucial differences are all localised to a solvent-exposed region in the N-terminal domain of TLP-ste. Ever since the unravelling of the three-dimensional structure of thermolysin in 1972 (Matthews et al., 1972) the structural characteristics underlying its extreme thermal stability have been the subject of speculation (Holmes & Matthews, 1982; Matthews et al., 1974; Stark et al., 1992). These characteristics have remained largely unsolved, despite the recent elucidation of the threedimensional structure of a less thermostable homologous proteinase produced by B. cereus (Stark et al., 1992). Thermolysin is the most thermostable member of a family of metallo-endopeptidases produced by various Bacillus species (these enzymes are also called 'neutral' proteinases or thermolysin-like proteinases, TLPs). Members of this family bind one zinc ion in their active site, and a varying number (2-4) of calcium ions that contribute to stability (Matthews et al., 1974; Dahlquist et al., 1976; Roche & Voordouw, 1978). TLPs consist of residues arranged with an amino-terminal domain predominantly consisting of $-strands and a carboxy-terminal domain which is mostly helical (Matthews et al., 1974; Stark et al., 1992; Figure 1). At elevated temperatures TLPs become irreversibly inactivated due to autolysis. TLPs have a broad proteolytic specificity (Heinrikson, 1977), thus conformational features rather than sequence characteristics determine the sites of autolytic attack (Fontana, 1988). It has been shown that the rate of thermal inactivation of TLPs is controlled by partial unfolding processes which render the molecule susceptible towards autolysis (Dahlquist et al., 1976; Van den Burg et al., 1990; Eijsink et al., 1991; Eijsink et al., 1992d; Braxton & Wells, 1992; Vriend & Eijsink, 1993; Hardy et al., 1994). TLPs exhibit large differences in stability to thermally induced autolysis and thus in their resistance against thermal unfolding. Thermal stability of the TLPs is quantified in terms of T 50, the temperature for which a 30 minute incubation reduces the enzyme activity by half. Thermolysin, whose T 50 is 86.9 EC, 90

4 Construction of extremely stable TLP-ste variants Figure 1 Ribbon drawing of the thermolysin-like proteinase of B. stearothermophilus (TLP-ste) showing the positions that were mutated (see Tables 1 and 2). The ribbon is coloured green at positions where mutations had an absolute effect on thermal stability of less than 1 EC. Yellow indicates positions where the absolute mutational effect was larger than 1 EC. The side chains of the six residues that were introduced in the most thermostable TLP-ste mutant obtained so far (Table 2) are shown in small spheres. The large spheres indicate the active site zinc (purple) and the four calcium ions (white) that are bound to the molecule. The active site zinc is located at the interface between the largely $-pleated N-terminal domain (right) and the largely "-helical C-terminal domain (left) of the molecule. differs from the less stable TLP from by B. stearothermophilus CU21 (T 50 = 73.4 EC) at 44 positions (Titani et al., 1972; Takagi et al., 1985). The overall fold of these enzymes is similar and all residues involved in catalysis and binding of metal ions are conserved. Using a protein engineering approach, we show here that the difference in thermal stability between thermolysin and TLP-ste is determined by only a small subset of the 44 mutations between the two enzymes. In particular we show that the thermal stability of TLP-ste is determined by a relatively small surfacelocated region in the N-terminal domain and that the enzyme can be stabilised dramatically by introduction of a few mutations in this region. These results reveal the structural characteristics underlying the high stability of thermolysin and indicate ways to introduced stabilisation of aspecific proteinases. 91

5 Chapter 4 Table 1 The effect of single mutations on the stability of the thermolysin-like proteinase from B. stearothermophilus (TLP-ste). Mutation a Replacement of residues by the corresponding residue in thermolysin 1 C"-H 2 O (C) C$-H 2 O (C) Accessible surface Ala2 Thr Ala4 Thr Gly44 Ala Arg45 Lys Asn46 Tyr Val49 Thr Thr56 Ala Gly58 Ala Thr63 Phe Ala69 Pro Gly89 Asn Gly141 Ala Arg182 Lys Ala201 Ser Thr232 Ile Cys288 Ala Asn311 Asp (C 2 ) *T 50 ( C) b Designed mutations at different positions in the C-terminal domain (residues ) 2 Ala193 Ser Ala167 Ser His216 Asn Asn238 Leu deletion of Gly264 Ala Val315 Ala deletion of 315, , , ,

6 Construction of extremely stable TLP-ste variants c Mutations in the hydrophobic core 3 Leu144 Phe Met168 Trp Met168 Ala Ala240 Val Ala241 Val Leu284 Trp Leu284 Ala Cys288 Leu Cys288 Ile Throughout this paper residues are numbered according to the sequence of thermolysin (Titani et al., 1972). The distance between a residue and the protein surface is illustrated by the columns C" - H 2 O and C$ - H 2 O, giving the shortest distance to a bulk water molecule of the alpha and beta carbon atom, respectively, in thermolysin (Vriend, 1990). The column 'accessible surface' gives the water accessible molecular surface area of the residue in thermolysin (Lee & Richards, 1971). The stretch is a solvent-exposed beta hairpin. All TLP-ste variants in this paper were similar to wild-type with respect to specific activity towards casein at 37 EC. Some of the data in this Table have been described previously in the indicated references. Detailed structural analyses of the mutations have been or will be published elsewhere. 1 Hardy et al., 1993; Van den Burg et al., Eijsink et al., 1992d; Vriend & Eijsink, Eijsink et al., 1992c. Residues crucial for TLP-ste stability Fifteen of the 44 differences between thermolysin and TLP-ste were selected for mutational analysis Of the substitutions concerning residues with largely identical structural environments in thermolysin and TLP-ste, those for which the thermolysin situation could be favourable for stability were selected. Such substitutions were selected on the basis of molecular modelling studies, using general concepts regarding protein stability for evaluation. They included the introduction of $- branched residues in a $-sheet (Ala2 Thr, Ala4 Thr), improvement of hydrogen bonding (Ala201 Ser) or a salt bridge (Asn311 Asp), and Gly Xxx or Xxx Pro mutations (Gly44- Arg45-Asn46 Ala-Lys-Tyr; Gly58 Ala, Ala69 Pro, Gly89 Asn, Gly141 Ala). The latter type of mutations was expected to be especially important because of their rigidifying effects on 93

7 Chapter 4 (partially) unfolded protein (Imanaka et al., 1986; Matthews et al., 1987; Hardy et al., 1993). Some substitutions were primarily selected on the basis of sequence comparisons with other TLPs (Van den Burg et al., 1991; Ala4 Thr, Thr56 Ala, Thr63 Phe). The substitutions Gly44- Arg45-Asn46 Ala-Lys-Tyr, Val49 Thr, Arg182 Lys, and Thr232 Ile were primarily selected because molecular modelling studies indicated that these could have relatively large effects. For these latter substitutions, the modelling studies could not provide clear indications about the expected effect on stability. The Cys288 Ala mutation creates a cavity in the interior of TLP-ste and was made in relation to a previous study on the contribution of the hydrophobic core to thermal stability (Eijsink et al., 1992c). The results of the mutations (Table 1a) show that only a few of the differences between the two enzymes affect thermal stability to a considerable extent. Furthermore, the results show that these differences mainly concern surface-located residues in the N-terminal domain of the molecule. Previously published mutagenesis studies of TLP-ste mainly concerned designed mutations in the C-terminal domain. The effects of these mutations were generally much smaller (Eijsink et al., 1992d; Vriend & Eijsink, 1993; Eijsink et al., 1992c) than the effects of some of the mutations in the N-terminal domain that are described here (compare Table 1a with 1b). Furthermore, the thermal stability of TLP-ste appeared to be remarkably insensitive for mutations in its hydrophobic core (Eijsink et al., 1992c; Table 1c). The general picture emerging from the present and previous studies is that the thermal stability of TLP-ste is determined by a relatively small cluster of surfacelocated residues in the $-pleated N-terminal domain (Figure 1). Bridging the stability gap To further investigate the difference in thermal stability between thermolysin and TLP-ste, multiple mutations were introduced in the latter enzyme. The combination of the stabilising mutations (listed in Table 1a) results in highly stable variants of TLP-ste. Combining the Thr63 Phe and Ala69 Pro mutations is sufficient to increase the T 50 of TLP-ste from 73.4 EC to 85.7 EC, thus almost bridging the difference in stability with thermolysin. One more mutation (either Thr56 Ala or Gly58 Ala) is sufficient to make TLP-ste more stable than thermolysin, and combining five different mutations in TLP-ste (Ala4 Thr, Thr56 Ala, Gly58 Ala, Thr63 Phe and Ala69 Pro) produces an enzyme with a T 50 of 93.3 EC. Remarkably, the stabilisation obtained in this fivefold TLP-ste mutant is considerably larger than the difference in thermal stability between thermolysin and TLP-ste. Clearly, evolution has not optimised thermolysin for stability and our observations lead to the striking conclusion that thermolysin could be stabilised considerably by replacing some of its residues by the corresponding residue found in the less thermostable TLP-ste. 94

8 Construction of extremely stable TLP-ste variants Figure 2 The location of Phe 63 and Pro 69 in thermolysin. The side chain of Phe 63 is partly solvent-exposed and in contact with the protruding side chains of four surface residues (Val 9, Arg 11, Gln 17, Gln 61). The side chain of Phe 63 covers an underlying $-pleated structure. Pro 69 is close to the surface but almost completely shielded from the solvent. Pro 69 is located in the somewhat irregular N-terminal turn of the only "-helix in the N-terminal domain that extends to residue 87 (Matthews et al., 1974). The helix following Pro 69 is largely buried, traversing the N-terminal domain. The results in Table 2 confirm that the extreme thermal stability of thermolysin, as compared to TLP-ste, is caused by a few mutations in the N-terminal domain. The most crucial residues seem to be the phenylalanine at position 63 and the proline at position 69 (Figure 2). The stabilising role of the partly solvent-exposed hydrophobic phenylalanine at position 63 is remarkable and not completely understood (Van den Burg et al., 1994). Additional mutations at position 63 have shown that hydrophobic interactions between the phenyl ring of Phe63 and the aliphatic parts of several protruding surface residues in its direct environment (Val9, Arg11, Gln17, Gln61) are important (Van den Burg et al., 1994; Figure 2). The stabilising effect of Pro69 is probably due to its rigidifying effect, which reduces unfolding and the flexibility of the partially unfolded structures that are possible substrates for autolysis (Matthews et al., 1987; Hardy et al., 1993). Mechanistic considerations 95

9 Chapter 4 The T 50 used to quantify thermal stability is a kinetic parameter, the magnitude of which is determined by the rate of partial unfolding processes that precede autolytic degradation. Studies of the autolytic process of broad-specificity proteinases (Dahlquist et al., 1976; Fontana, 1988; Braxton & Wells, 1992) and observations concerning the structural changes during protein unfolding (Matoushek et al., 1989; Jackson & Fersht, 1991) suggest that these unfolding processes involve solvent-exposed regions of the protein. The present data support this by showing that only mutations at surface-located positions have large effects on thermal stability. Furthermore, the present data illustrate the partial character of the stability-determining unfolding processes, by showing that crucial mutations are clustered in a relatively small region of the protein. Most mutations outside this critical region have marginal effects on thermal stability (Table 1; Figure 1). The present results show that the region around residues 63 and 69 is involved in early steps of the thermal unfolding of TLP-ste and they suggest that this region is the first to reach autolytically susceptible conformations upon heating. The conclusion that the N-terminal domain is involved in the early steps of thermal unfolding is in excellent agreement with the results of previous studies on the folding, unfolding and stability of thermolysin and fragments thereof. These studies showed that the N-terminal and C-terminal domain have different intrinsic stabilities and that the N-terminal domain is relatively unstable (Corbett et al., 1986; Vita et al., 1989). The precise character of the rate-limiting unfolding and subsequent autolysis processes affected by the critical mutations identified in this study is, however, largely unknown. This is partly due to the fact that so far no kinetically relevant autolytic breakdown products of TLP-ste have been isolated. Attempts to isolate such breakdown products are hampered by the fact that autolysis proceeds very rapidly after the first rate-limiting step (Van den Burg et al., 1990; Eijsink et al., 1991b; and V. G. H. Eijsink, B. van der Vinne & G. Venema, unpublished results). Because of the obscurity of the crucial autolysis sites it is difficult to estimate the extent of the partial unfolding processes that are affected by the stabilising mutations. It can not be excluded that these mutations affect the unfolding of surface loops distant from the mutation sites, and, consequently, the crucial cleavage site may be located outside the direct environment of the mutated residues. Recent modelling studies of the change in conformation required for limited proteolytic cleavage of a protein indicate that autolysis in any case requires a considerable degree of unfolding at the actual cleavage site, most probably involving at least 12 residues (Hubbard et al., 1994). In one of the early papers concerning the conformation of thermolysin it was stated that the binding of four calcium ions was the only noticeable structural feature to which the extreme stability of this protein could be attributed (Matthews et al., 1974; Figure 1). Studies on the contribution of these calcium ions to thermolysin stability (Dahlquist et al., 1976; Roche & Voordouw, 1978; Fontana, 1988) showed that during thermal inactivation one calcium ion was released from the 96

10 Construction of extremely stable TLP-ste variants molecule. The present data suggest that this calcium must be the one co-ordinated by Asp57 and Asp59, located in the thermal stability-determining region. Replacing Asp57 by serine resulted in a destabilisation of approximately 7 EC, in both wild-type TLP-ste and the Phe63, Pro69 twofold mutant of this enzyme (F. Hardy, G. Vriend, O. R. Veltman; G. Venema & V. G. H. Eijsink, unpublished data). Thus, binding of calcium seems to make a significant, but not predominant contribution to thermal stability. Table 2 Engineering extremely thermal stable variants of the thermolysin-like proteinase of B. stearothermophilus (TLP-ste) TLP variant T 50 ( C) *T 50 ( C) TLP-ste wild-type 73.4 T63F + A69P Thermolysin (44 mutations) T56A + T63F + A69P T58A + T63F + A69P T56A + G58A + T63F + A69P A4T + T56A + G58A + T63F + A69P T56A + G58A + T63F + A69P + S65P A4T + T56A + G58A + T63F + A69P + S65P The stability of the enzymes is given as their T 50 and as their *T 50 compared to wild-type TLP-ste. The error margins in both types of values are approximately 0.4 EC. All individual mutations concern the replacement of a residue by the residue that occurs at the corresponding position in thermolysin, except S65P. The latter mutation was designed on the basis of general principles concerning protein stability (Hardy et al., 1993). Commercially available thermolysin was purified with the methods used for the TLP-ste variants, before determining its thermal stability. 97

11 Chapter 4 Engineering extremely stable TLPs The identification of regions that unfold relatively easy to proteolytically susceptible conformations is crucial for the design of stabilising mutations in aspecific proteinases. More generally, the identification of such regions is critical for the design of mutations aimed at stabilising any protein against proteolytic attack. Mutations aimed at reducing unfolding and subsequent proteolysis only have noticeable effects on thermal stability if they are introduced in a previously identified critical region, such as the region around residues 63 and 69 in TLP-ste. This is illustrated by for example the designed Ser65 Pro mutation in TLP-ste: the introduction of this mutation in wild-type TLPste appeared to be very effective and resulted in a stabilisation of 4.7 EC (Hardy et al., 1993). Another example is a large stabilisation that was obtained recently by introducing a disulphide bridge in the critical region of TLP-ste (J. Mansfeld, G. Vriend, B. W. Dijkstra, O. R. Veltman, B van den Burg, G. Venema, R. Ulbrich-Hofmann & V. G. H. Eijsink, manuscript in preparation). The latter result contrasts with the lack of success of several previous studies aimed at stabilising TLPs or other broad-specificity proteinases (subtilisins) by the introduction of disulphide bridges (see Van den Burg et al., 1993 and references therein). This lack of success was most probably caused by the fact that the disulphide bridges were introduced in parts of the protein that do not play a role in the partial unfolding processes that determine thermal stability. The importance of finding the best target regions for stabilising mutations is amply illustrated by the result of introducing the Ser65 Pro mutation in the fivefold mutant of TLP-ste. This results in one of the most stable proteins ever obtained by protein engineering (T 50 = 96.9 EC; Table 2, Figure 3a). The extreme stability of this sixfold mutant is illustrated by Figure 3b: the mutations increase the half-life of TLP-ste at 90 EC more than 100-fold, from less than one minute to 118 minutes. The sixfold mutant is much more stable than thermolysin, which has a half-life of only six minutes at 90 EC. The present data show that the thermal stability of TLP-ste can be increased dramatically by just a few mutations in one particular region of the protein. Our observations seem to disagree with observations by Serrano et al. (1993) who showed that most of the 17 substitutions between two ribonucleases with 85 % sequence identity had comparable modest effects on thermodynamic stability (resistance to global unfolding) and that the stability determining substitutions were scattered over the molecule. This seeming contrast is most likely caused by the fact that TLP stability is determined by partial (as opposed to global) unfolding processes. This makes that in TLPs only mutations in regions that unfold relatively easily to autolytically susceptible conformations have noticeable effects on thermal stability. The conclusion drawn by Serrano et al. (1993) that there is no clear correlation between the type of mutation and the effect 98

12 Construction of extremely stable TLP-ste variants Figure 3 a, Determination of T 50 for TLP-ste wild-type (+), thermolysin (Î), TLP-ste with mutations A4T, T56A, G58A, T63F, A69P ( ), TLP-ste with mutations A4T, T56A, G58A, T63F, S65P, A69P (+). b, Stability of TLPste wild-type (+), thermolysin (Î), and TLP-ste with mutations A4T, T56A, G58A, T63F, S65P, A69P ( ) at 90 EC. on protein stability is confirmed by the present study. The substitutions critical for TLP thermal stability differ in character (although rigidification seems to be some kind of a common denominator) and many of them are not directly obvious from inspections and comparisons of the primary and tertiary structures of thermolysin and its less stable counterparts (Matthews et al., 1974; Holmes & Matthews, 1982; Stark et al., 1992). As already noted in 1974, 'there is nothing particularly unusual in the thermolysin conformation to which one might attribute the thermostability of the molecule' (Matthews et al., 1974). The present results illustrate this by showing that extreme stability can be caused by very few mutations which are not always predictable on the basis of present concepts regarding protein stability. 99

13 Chapter 4 METHODS Production of mutant enzymes. The npr gene encoding TLP-ste was cloned (Fujii et al., 1983), sequenced (Takagi et al., 1986), and subcloned (Eijsink et al., 1992c) as described previously. All methods for sitedirected mutagenesis, gene expression, and production and purification of TLP-ste variants have been described elsewhere (see Van den Burg et al., 1990 and references therein). Multiple mutations were introduced by successive mutagenesis steps and by combining appropriate restriction fragments of individually mutated TLP-ste genes. Characterisation of TLPs. Thermal stability was quantified as T 50, being the temperature of incubation at which 50 % of the initial activity of a 0.1 :M solution of purified enzyme [in 20 mm sodium acetate, ph 5.3, 5 mm CaCl 2, 0.5 % (v/v) isopropanol, 62.5 mm NaCl] is retained in 30 minutes. Proteinase activity was determined using a casein assay, at 37 EC (Fujii et al., 1983). The stability of the mutant enzymes is expressed as *T 50, being the difference in T 50 between the wild-type (T 50 = 73.4 EC) and the mutant enzyme. The error margins in the *T 50 values are estimated to be EC, depending on the magnitude of *T 50. T 50 and *T 50 values given here differ from previously published values because mercury thermometers were used for temperature registration instead of the previously used alcohol thermometers. The latter were found to be insufficiently accurate at the extreme temperatures employed in the present study. For some TLPs the stability over time at 90 EC was determined under conditions identical to those used for the determination of T 50. Aliquots were removed from the incubation bath after various periods of time and assayed for remaining activity towards casein at 37 EC. Structural analysis and molecular modelling. A model of TLP-ste was built-by-homology (85% sequence identity) on the basis of the crystal structure of thermolysin (Matthews et al., 1972; Holmes & Matthews, 1982). Three extra residues present in TLP-ste (inserted in the region; Titani et al., 1972; Takagi et al., 1985) could not be modelled accurately and were omitted from the model. The model-building studies have indicated that TLP-ste is structurally very similar to thermolysin (including the bound metal ions) and the structural environment of the residues listed in Table 1 is expected to be almost identical in both enzymes. All model-building procedures were performed using the program WHAT IF (Vriend, 1990) and have been described elsewhere (Vriend & Eijsink, 1993). The program for drawing the ribbon is described in (Carson, 1987). Throughout this paper residues are numbered according to the sequence of thermolysin (Titani et al., 1972). 100

14 Construction of extremely stable TLP-ste variants Acknowledgements We thank B. van der Vinne and F. Hardy for their help with some of the experiments, H. Mulder for preparing some of the pictures, T. Imanaka for sending us his plasmids pnp22 and pnp28, and I. F. Nes for general support. REFERENCES References are listed in chapter

15 Chapter 4 102

16 Construction of extremely stable TLP-ste variants 103