Increased thermal stability against irreversible inactivation of 3-isopropylmalate dehydrogenase induced by decreased van der Waals volume at the subunit interface

Takatoshi Ohkuri and Akihiko Yamagishi1

Department of Molecular Biology, Tokyo University of Pharmacy and Life Science, 1432 Horinouchi, Hachioji, Tokyo 192-0392, Japan

1 To whom correspondence should be addressed. e-mail: yamagish{at}ls.toyaku.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We have investigated factors affecting stability at the subunit–subunit interface of the dimeric enzyme 3-isopropylmalate dehydrogenase (IPMDH) from Bacillus subtilis. Site-directed mutagenesis was used to replace methionine 256, a key residue in the subunit interaction, with other amino acids. Thermal stability against irreversible inactivation of the mutated enzymes was examined by analyzing the residual activity after heat treatment. The mutations M256V and M256A increased thermostability by 2.0 and 6.0°C, respectively, whereas the mutations M256L and M256I had no effect. Thermostability of the M256F mutated enzyme was 4.0°C lower than that of the wild-type enzyme. To our surprise, increasing the hydrophobicity of residue 256 within the hydrophobic core of the enzyme resulted in a lower thermal stability. The mutated enzymes showed an inverse correlation between thermostability and the volume of the side chain at position 256. Based on the X-ray crystallographic structure of Escherichia coli IPMDH, the environment around M256 in the B.subtilis homolog is predicted to be sterically crowded. These results suggest that Met256 prevents favorable packing. Introduction of a smaller amino acid at position 256 improves the packing and stabilizes the dimeric structure of IPMDH. The van der Waals volume of the amino acid residue at the hydrophobic subunit interface is an important factor for maintaining the stability of the subunit–subunit interface and is not always optimized in the mesophilic IPMDH enzyme.

Keywords: dimeric enzyme/hydrophobic core/isopropylmalate dehydrogenase/subunit interaction/thermostability


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein–protein interactions are essential for many biological processes including polypeptide oligomerization, gene expression, replication, signal transduction, enzyme regulation and the immune response (Nossal, 1992Go; Zhou and Cantley, 1995Go; Davies and Cohen, 1996Go; Darnell, 1997Go; Ptashne and Gann, 1997Go). A better understanding of the principles of protein–protein interaction is essential in order to establish the underlying molecular mechanisms of these biological processes. Proteins of high molecular mass tend to have multi-subunit entities, and homo-dimerization is a common property of many proteins (Darnall and Klotz, 1975Go). The subunit interaction in a dimeric enzyme can be a model for understanding protein–protein interactions. The dimer interface is maintained by hydrophobic interaction, as well as by hydrogen bonding and the presence of salt bridges at the interface (Chothia and Janin, 1975Go; Kabsch and Sander, 1983Go; Horton and Lewis, 1992Go; Bashir et al., 1995Go). The basic properties of the subunit–subunit interface are expected to be the same as those responsible for the folding of monomeric proteins (Walls and Sternberg, 1992Go; Young et al., 1994Go; Tsai et al., 1996Go). However, the mechanism of formation of dimeric structures is not as well understood as those of protein folding.

3-Isopropylmalate dehydrogenase (IPMDH) is a homodimeric enzyme of the leucine biosynthetic pathway. Genes encoding this enzyme have been cloned and sequenced from a variety of sources. The three-dimensional structure of the IPMDH from an extreme thermophile, Thermus thermophilus (TtIPMDH), showed it to be composed of two identical subunits. The subunit–subunit interaction of IPMDH is maintained by the four-helix bundle formed by the two helices of each subunit and the hydrophobic core of the bundle composed of some hydrophobic residues (Imada et al., 1991Go). The hydrophobic region at the subunit interface was highly conserved between the thermophile and mesophile IPMDHs from Escherichia coli (EcIPMDH) and Bacillus subtilis (BsIPMDH). However, Leu246 and Val249 in T.thermophilus are Glu and Met, respectively, in the corresponding mesophilic enzymes. When Glu256 of EcIPMDH and Met259 of EcIPMDH in the hydrophobic core were substituted with Leu and Val, respectively, which are the corresponding residues in TtIPMDH, thermostability increased (Kirino et al., 1994Go). Mutated B.subtilis IPMDH (Glu253Leu or Met256Val) also displayed elevated thermal stability compared with B.subtilis IPMDH without these mutations (Akanuma et al., 1999Go). These results were interpreted as meaning that hydrophobic interactions at the subunit interface are critically important for the thermostability of dimeric enzymes. In this study, we have examined the role of Met256 in the hydrophobic core of the subunit interface of BsIPMDH by mutating this position and analyzing the thermal stability of the resulting proteins.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Materials

3-Isopropylmalate and NAD were purchased from Wako Pure Chemicals and Orient Yeast Ltd, respectively (Tokyo, Japan). All other reagents were of analytical grade.

Construction of mutated enzymes.

Site-directed mutagenesis was carried out by PCR according to the methods of Picard et al. (Picard et al., 1994Go). Plasmid pET21c, which harbors the gene for BsIPMDH, was used as the template. The sequences of the flanking primers used for mutagenic PCR were 5'-TAATACGACTCACTATAGGG-3' and 5'-CTAGTTATTGCTCAGCGGT-3'. The primers used for mutagenesis were 5'-TAAGCGATGAAGCGTCCSY TCTTACAGGCTCGCTCGG-3' for substitution of Met256 to Ala, Val or Phe, and 5'-TAAGCGATGAAGCGTCCM TCCTTACAGGCTCGCTCGG-3' for substitution of Met 256 to Ile or Leu. Mutations were confirmed by sequence analysis (Sanger et al., 1977Go).

Enzyme purification

Expression and purification of the wild-type and mutated IPMDHs were carried out as described previously (Hayashi-Iwasaki and Oshima, 2000Go). Wild-type and mutant IPMDHs were overexpressed in E.coli BL21 ({Delta}leuB) cells harboring the recombinant plasmid constructed using a pET21c expression vector. Cells were harvested and disrupted by sonication and the cell-free extract was clarified by centrifugation. The enzyme was purified by standard column chromatography (Hayashi-Iwasaki and Oshima, 2000Go), using Hiprep Q (Amersham Pharmacia Biotech, Uppsala, Sweden), Phenyl Toyopearl (Toso, Tokyo, Japan) and Resource Q (Amersham Pharmacia Biotech) columns.

Enzyme assay

The enzyme activity was measured in 100 mM potassium phosphate buffer (pH 7.6), containing 1 M KCl, 0.2 mM MnCl2, 0.8 mM D,L-isopropylmalate and 0.8 mM NAD, by monitoring the absorbance at 340 nm, using a Beckman DU7400 spectrophotometer, at 40°C, according to the method described previously (Yamada et al., 1990Go).

Measurement of thermal stability against irreversible inactivation

The residual activity of enzyme after heat treatment was estimated according to the previously described method (Kotsuka et al., 1996Go). The enzyme was diluted to 0.4 mg/ml with 20 mM potassium phosphate (pH 7.6), containing 0.5 mM EDTA, and incubated at various temperatures for 10 min. After the heat treatment, the samples were immediately cooled on ice for 10 min and centrifuged at 21 000 g for 10 min. The residual activity of the supernatant was measured at 40°C. Thermal denaturation was monitored by circular dichroism (CD) at 222 nm with a Jasco J-720C spectropolarimeter (Iwasaki et al., 1996Go). The enzyme was dissolved (0.2 mg/ml) in 20 mM potassium phosphate (pH 7.6). The temperature of the enzyme solution was controlled with a circulating bath and increased with a programmable temperature controller (Neslab; Thermo Electron Co., Waltham, MA) at a scan rate of 1.0°C/min. The temperature of the sample was monitored with a thermocouple.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Thermal stabilities against irreversible inactivation of mutant BsIPMDHs

To examine the relationship between hydrophobicity of residue 256 and the thermostability of BsIPMDH, we prepared the mutant enzyme by site-directed mutagenesis in which Met256 was substituted with Ala, Val, Leu, Ile or Phe. Mutated forms of BsIPMDH were overexpressed in E.coli BL21 ({Delta}leuB) cells and purified to homogeneity.

The thermal stability of the purified mutated BsIPMDHs were estimated by measuring the residual activities at 40°C after heat treatment at various temperatures for 10 min. The thermal inactivation processes of the wild-type and mutant BsIPMDHs were irreversible under the conditions used. Figure 1 shows the residual activities after heat treatment. The specific activity of all non-heat-treated mutant BsIPMDHs did not differ from that of the wild-type BsIPMDH by >5% (data not shown). The Th (temperature at which half of the protein is denatured) of BsIPMDH-M256V was 2°C higher than that of wild-type BsIPMDH, but was unchanged for BsIPMDH-M256L and BsIPMDH-M256I despite the substitution of more hydrophobic residues. Furthermore, the Th of BsIPMDH-M256F was 4.5°C lower than that of the wild-type BsIPMDH. In contrast, BsIPMDH-M256A had a Th 6.0°C higher than that of wild-type BsIPMDH. These results indicate that the effect of hydrophobicity at position 256 on the thermal stability is contrary to expectations.



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Fig. 1. Thermal stability against irreversible inactivation of M256 mutant IPMDHs. Residual activity was assayed after the heat treatment at the indicated temperature for 10 min.

 
The thermal denaturation of wild-type and mutant BsIPMDHs was analyzed by measuring the change in ellipticity at 222 nm (Figure 2). Thermal denaturation processes of wild-type and mutant BsIPMDHs were irreversible under the conditions used. Some of the mutant BsIPMDHs, especially BsIPMDH-M256F, showed two-phase denaturation curves. No deviation of the denaturation curve was detected by increasing the concentration of BsIPMDHs to 0.4 mg/ml. Tm values (temperature at which 50% the original secondary structure is lost) of the mutant BsIPMDHs were compared with that of wild-type BsIPMDH. BsIPMDH-M256V and BsIPMDH-M256A had Tm values that were 2.5 and 7.0°C higher than that of wild-type BsIPMDH, respectively. The Tm values of BsIPMDH-M256I and BsIPMDH-M256L were similar to that of wild-type BsIPMDH. However, the Tm of BsIPMDH-M256F was 12.5°C lower than that of wild-type BsIPMDH.



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Fig. 2. Thermal melting profiles of the M256 mutant IPMDHs. Thermal denaturation was monitored by recording the change in CD at 222 nm. The enzymes were dissolved in 20 mM potassium phosphate (pH 7.6). The enzyme concentration was 0.2 mg/ml.

 
Relationship between thermostability and the volume of the amino acid residue at position 256

If the hydrophobicity of the amino acid residue at position 256 is the principal factor governing thermostability, we would predict that IPMDHs with a more hydrophobic residue (BsIPMDH-M256I, -M256L and -M256F) should have an elevated Tm whereas the mutated form with a less hydrophobic residue (BsIPMDH-M256A) would have a lower Tm. Thus, our experimental findings are contrary to predictions. An increase in thermostability was detected for mutated proteins where a smaller amino acid residue, such as Ala or Val, was substituted at position 256. We examined the relationship between Th and Tm against the van der Waals volume of the amino acid residue 256 (Figure 3). The Th or Tm decreased depending on the increase in the van der Waals volume of the amino acid residue at position 256. The Tm of the M256F variant deviated from the Th, but the Tm of the second phase of the denaturation curve was similar to the Th.



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Fig. 3. Correlation between Th (a) and Tm (b) of the M256 mutant IPMDHs and the van der Waals volume of the amino acid residue at position 256. The van der Waals volume of the amino acid residue was estimated by Richards (Richards, 1974Go). The Tm of Phe* indicates the temperature of the second phase of the denaturation curve of BsIPMDH-M256F.

 
Although the structure of BsIPMDH is not known, the atomic structures of TtIPMDH and EcIPMDH have been reported and were found to be very similar to each other (Imada et al., 1991Go; Wallon et al., 1997aGo). Since the primary amino acid sequence of BsIPMDH is closely related to that of TtIPMDH and EsIPMDH, its three-dimensional structure is also likely to be similar (Numata et al., 1995Go; Akanuma et al., 1999Go).

Figure 4 shows the side view of the dimer interface which is composed of hydrophobic residues from four helices; two helices from each subunit. Figure 5 shows the bottom view of the four-helix bundle at the interface. In TtIPMDH, Val224 residues from two subunits are located at the center of the four-helix bundle. Val224 and Val224' are sandwiched between Val249 and Val249'. Although Val249 and Val249' are located close to the outer surface of the enzyme, they are almost buried, having a solute-accessible surface area of only 3.1%. The corresponding residues in EcIPMDH have a similar conformation in which Ile234 and Ile234' (Ile231 and Ile231' of BsIPMDH) are surrounded by Met259 and Met259' (Met256 and Met256' of BsIPMDH). The Met259 residue in EcIPMDH is still buried and has a solvent-accessible area of 3.1%. In this study, proteins with a smaller amino acid residue at position 256 of BsIPMDH (EcIPMDH-M259) showed increased thermal stability against irreversible inactivation, indicating an inverse relationship between the enzyme’s thermostability and the van der Waals volume of the amino acid residue.



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Fig. 4. The subunit interface with the four-helix bundle formed by two helices from each subunit of IPMDH from T.thermophilus (A) and E.coli (B). The residues involved in the subunit interaction at the hydrophobic core are shown. Residues in each subunit are shown either in green or blue. Residues shown in ball-and-stick model are Val224 [darker colors in (A)], Val249 [lighter colors in (A)], Ile234 (Ile231 of BsIPMDH) [darker colors in (B)] and Met259 (Met256 of BsIPMDH) [lighter colors in (B)]. The figure was drawn using InsightII.

 


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Fig. 5. The structures of TtIPMDH (A) and EcIPMDH (B) around the residue corresponding to M256 of BsIPMDH. A prime indicates the residues from the other subunit. The coordinates of the hydrogen atoms are not known. The figure was drawn using InsightII.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Based on crystal structure analysis of IPMDH, hydrophobic interactions at the subunit interface were reported to be crucial for protein stabilization (Imada et al., 1991Go). Two residues, found in the hydrophobic core of TtIPMDH, were substituted in EcIPMDH and BsIPMDH for the more hydrophobic residues, Leu246 and Val249. EcIPMDH carrying E256L and M259V mutations showed higher thermal stability than wild-type EcIPMDH (Kirino et al., 1994Go). BsIPMDHs carrying the E253L or M256V mutation also displayed enhanced thermostability (Akanuma et al., 1999Go). These results have been interpreted as meaning that an increase in hydrophobicity at two positions in the hydrophobic core of the protein enhances the stability of the dimeric enzyme.

In this study using BsIPMDH, we found that the single-mutation M256V increased thermostability over the wild-type enzyme, supporting the previous interpretation. However, as shown in Figure 3, hydrophobicity of the amino acid residue at position 256 was inversely correlated to the thermal stability against irreversible inactivation of BsIPMDH. A mutated enzyme carrying a smaller amino acid residue at position 256 showed higher thermostability.

Denaturation of wild-type BsIPMDH was irreversible. Accordingly, the denaturation curve represents the denatured form accumulated during the gradual increase in temperature during the CD measurement. However, residual activity was estimated after the heat treatment for 10 min [a 10 min denaturation has been used for the estimation of the thermal stability of IPMDHs for the other organisms (Yamada et al., 1990Go; Numata et al., 1995Go; Yoda et al., 1995Go; Wallon et al., 1997Gob; Tamakoshi et al., 2001Go)]. The residual activity is expected to be directly proportional to the fraction of native enzyme after the 10 min heat treatment. Thus, the Th estimated by residual activity does not necessarily coincide with the Tm estimated by CD measurement. However, the temperatures estimated by these two methods were similar for each mutated protein (Figure 3).

A two-phase denaturation curve was observed in the CD measurement of BsIPMDH-M256F (Figure 2). It took 10 min to increase the temperature of the protein solution from 30 to 40°C. Thus, the first phase of the denaturation process, monitored by CD, was expected to be complete after treatment at 40°C for 10 min. However, when the residual activity of BsIPMDH-M256F after the 10 min heat treatment was estimated (Figure 1), we observed only a 10% decrease in activity. Therefore, the first phase of the BsIPMDH-M256F denaturation curve measured by CD appears to be reversible or at least unrelated to the loss of activity. Though detailed analysis may be necessary to elucidate the characteristics of each phase of the denaturation curve, the Tm of the second phase was rather closer to the Th estimated by the residual activity in BsIPMDH-M256F.

Figure 4 shows the side view of the helix-bundle structures of TtIPMDH and EcIPMDH observed in the plane of the subunit–subunit interface. From this perspective, the distance between the two helices of the different subunits is even from top to bottom in EcIPMDH while the distance is shorter at the bottom in TtIPMDH. The corresponding amino acid residues at the bottom of the helices are closer in TtIPMDH than EcIPMDH: the distance between the {alpha} carbons of Val249 and Val224' is 7.4 Å in TtIPMDH, while that of Met259 and Ile234' is 7.7 Å in EcIPMDH.

In the structure of TtIPMDH, the distance between the {gamma} carbon atom of V249 and the {gamma} carbon atom of V224' is 4.0 Å (Figure 5A). Val249 is also surrounded and sandwiched by Met221' and Arg225' residues from the top and from the bottom of Val249, respectively (not shown in Figure 5). In the structure of EcIPMDH, Met259 and Ile234' are also in close contact: the distance between the {gamma} carbon of Met259 and the {gamma}2 carbon of Ile234' is 4.0 Å. The Met259 residue is also surrounded and sandwiched by Met231' and Lys235' residues. Although still buried, the sulfur atom and {epsilon} carbon of Met259 project outwards. Thus, the total volume occupied by the amino acid residues in this region in EcIPMDH is larger than that in TtIPMDH. The combination of these residues appears to determine the distance between the two helices in these proteins. Substitution of residues with a larger van der Waals volume may have introduced stress in the conformation of side chains at the subunit interface. Alternatively for EcIPMDH, the bulky residues in this region may have increased the distance between the helices, thereby affecting the interaction immediately above these residues.

Table I shows residues corresponding to I231 and M256 of BsIPMDH in IPMDHs from other microorganisms. Combinations of the corresponding residues are different in different organisms. Although different, none of the combinations of two residues have large volumes except for BsIPMDH and EcIPMDH. Interestingly, for the ultrathermophile Thermotoga maritima, there are hydrophobic residues at the two corresponding sites (I231 and A256). This combination is the same as that of BsIPMDH-M256A that showed the highest thermal stability among the BsIPMDH-M256 mutants tested.


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Table I. Residues corresponding to I231 and M256 of BsIPMDH in IPMDH from various microorganisms
 
An alternative interpretation of the impact of these mutations on protein structure is that exposure of these hydrophobic residues to water may cause instability. This is unlikely for residues the same size as or smaller than Met, because Met259 in EcIPMDH has a solute-accessible surface area of only 3.1%. However, in BsIPMDH-M256F the Phe residue occupies a relatively large volume and may indeed destabilize the enzyme by exposure to water.

Glaser et al. determined the residue–residue preferences in intermolecular interactions based on a database of numerous co-crystallized protein–protein interfaces (Glaser et al., 2001Go). Their results revealed that the highest residue–residue preferences at the interface were for interactions between pairs of large hydrophobic residues, and the lowest preferences for interactions between pairs of small residues. Experimental studies by Vallone et al. have indicated that there is a correlation between the change in the free energy of association of the mutants and the change in buried hydrophobic surface area (Vallone et al., 1998Go). Thus, the hydrophobicity at the subunit interface is expected to be the main driving force for protein–protein interactions.

Nevertheless, hydrophobic packing, rather than hydrophobicity alone, at the hydrophobic core of the protein interior, is essential for the stability of the native conformation of proteins. Lim and Sauer suggested that the total volume and steric interaction of residues in the hydrophobic core is the limiting factor for protein stability based on their experiment involving random mutagenesis (Lim and Sauer, 1989Go). In this study, we found that the volume of an amino acid residue in the hydrophobic core at the subunit interface is also important for the stability of a dimeric protein. In conclusion, hydrophobic packing, rather than hydrophobicity at the interface, appears to be a crucial factor in protein–protein interaction.


    Acknowledgements
 
This work was supported by the ACT-JST and BIRD-JST programs from the Japan Science and Technology Corporation.


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received September 3, 2002; revised June 13, 2003; accepted June 20, 2003.





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