University of Groningen

Engineering high performance variants of Bacillusthermolysin-like proteasesVeltman, Oene Robert

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Construction of extremely stable TLP-ste variants

89

Chapter 4

Structural Determinants Of The Stability Of

Thermolysin-Like Proteinases

Published in: Nature Structural Biology (1995) 2, 374-379.

Chapter 4

90

SUMMARY

Thermolysin is a member of a family of homologous proteinases which differ in their

resistance to thermally induced unfolding and subsequent autolytic degradation. Site-

directed 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 three-

dimensional 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 300-319 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 T50, the temperature for which a 30 minute

incubation reduces the enzyme activity by half. Thermolysin , whose T50 is 86.9 EC,

Construction of extremely stable TLP-ste variants

91

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 (T50 = 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 surface-

located 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.

Chapter 4

92

Table 1 The effect of single mutations on the stability of the thermolysin-like proteinase from B.

stearothermophilus (TLP-ste).

Mutation C"-H2O

(C)

C$-H2O

(C)

Accessible surface

(C2)

*T50(°C)

a Replacement of residues by the

corresponding residue in thermolysin1

Ala2 → Thr 3.4 3.3 37 - 0.4

Ala4 → Thr 3.6 3.2 21 + 1.9

Gly44 Ala 6.1 6.9 0.8Arg45 →Lys 4.2 3.3 35 - 0.2Asn46 Tyr 5.0 4.1 31Val49 → Thr 3.3 3.6 29 - 0.3

Thr56 → Ala 4.6 4.0 13 + 1.9

Gly58 → Ala 3.8 3.3 22 + 3.9

Thr63 → Phe 4.0 3.3 26 + 7.0

Ala69 → Pro 6.3 5.5 1.6 + 6.3

Gly89 → Asn 4.4 3.2 38 - 0.3

Gly141→ Ala 11.8 10.8 0 + 0.7

Arg182→ Lys 3.4 3.5 13 - 2.9

Ala201→ Ser 6.5 6.0 0.8 - 0.4

Thr232→ Ile 5.4 5.1 13 -0.6

Cys288→ Ala 8.6 9.0 0 0.0

Asn311→ Asp 4.2 3.5 19 - 0.3

b Designed mutations at different positions in

the C-terminal domain (residues 155-316)2

Ala193→ Ser 9.1 8.4 0 + 0.9

Ala167→ Ser 12.7 13.2 0 + 0.8

His216→ Asn 6.6 5.1 12 0.0

Asn238→ Leu 11.1 11.0 0.2 + 0.8

deletion of 248-257 0.0

Gly264→ Ala 8.4 8.7 1.0 + 0.2

Val315→ Ala 5.4 6.8 1.0 0.0

deletion of 315,316 5.4, 3.6 6.8, 3.3 1.0, 42 - 0.3

Construction of extremely stable TLP-ste variants

93

c Mutations in the hydrophobic core3

Leu144→ Phe 11.6 12.2 0.7 0.0

Met168→ Trp 14.0 12.8 0.2 + 0.3

Met168→ Ala 14.0 12.8 0.2 - 0.1

Ala240→ Val 11.2 12.4 0 0.0

Ala241→ Val 11.7 12.3 0 - 0.1

Leu284→ Trp 7.8 9.1 0 + 0.3

Leu284→ Ala 7.8 9.1 0 0.0

Cys288→ Leu 8.6 9.0 0 + 0.2

Cys288→ Ile 8.6 9.0 0 + 0.4

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" - H2O and C$ - H2O, 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 248-257 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., 1991.2 Eijsink et al., 1992d; Vriend & Eijsink, 1993.3 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

Chapter 4

94

(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 surface-

located 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 T50 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 T50 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.

Construction of extremely stable TLP-ste variants

95

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

Chapter 4

96

The T50 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

Construction of extremely stable TLP-ste variants

97

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 T50 (°C) *T50 (°C)

TLP-ste wild-type 73.4

T63F + A69P 85.7 + 12.3

Thermolysin (44 mutations) 86.9 + 13.5

T56A + T63F + A69P 87.8 + 14.4

T58A + T63F + A69P 89.1 + 15.7

T56A + G58A + T63F + A69P 92.2 + 18.8

A4T + T56A + G58A + T63F + A69P 93.3 + 19.9

T56A + G58A + T63F + A69P + S65P 95.1 + 21.7

A4T + T56A + G58A + T63F + A69P + S65P 96.6 + 23.5

The stability of the enzymes is given as their T50 and as their *T50 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.

Chapter 4

98

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 TLP-

ste 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 (T50 = 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

Construction of extremely stable TLP-ste variants

99

Figure 3 a, Determination of T50 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 TLP-

ste 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.

Chapter 4

100

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 site-

directed 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 T50, 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 CaCl2, 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 *T50, being the difference in T50 between the wild-type (T50 = 73.4 EC) and the mutant enzyme.

The error margins in the *T50 values are estimated to be 0.2 - 0.4 EC, depending on the magnitude of *T50.

T50 and *T50 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

T50. 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 25-30 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).

Construction of extremely stable TLP-ste variants

101

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 9.

Chapter 4

102

Construction of extremely stable TLP-ste variants

103