Tolerance of point substitution of methionine for isoleucine in hen egg white lysozyme

Tadahiro Ohmura, Tadashi Ueda, Yoshio Hashimoto and Taiji Imoto1,

Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan


    Abstract
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X-ray structure determination of proteins by using the multiple-wavelength anomalous dispersion method targeting selenomethionine is now widely employed. Isoleucine was examined for the second choice of the `safe' substitution of methionine next to leucine. We performed a systematic mutational study of the substitutions of methionine for isoleucine. All mutated lysozymes were less stable than the wild-type by about 1 kcal/mol and it is suggested that this instability was caused by the change in residual hydrophobicity from isoleucine to methionine. The X-ray structures of all mutant lysozymes were very similar to that of the wild-type. In addition, both the accessible surface areas and the conformation of the side chain of methionine in all mutant lysozymes were similar to those of the side chain at the respective isoleucine in the wild-type. Therefore, it is suggested that the mutation from isoleucine to methionine in a protein can be considered as a `safe' substitution.

Keywords: hen egg white lysozyme/isoleucine/`safe' substitution/selenomethionine/X-ray crystallography


    Introduction
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 Introduction
 Materials and methods
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The crystallographic study of a protein is indispensable for elucidating its structure and function. Recently, X-ray crystallography of a protein using synchrotron orbit radiation has been developing rapidly. This approach has several merits: one can collect the reflection data in the case of very small crystals because the synchrotron orbit radiation has high energy; in addition, one can determine the novel structure without heavy atom isomorphous crystals by using the multiple-wavelength anomalous dispersion method targeting selenomethionine (Hendrickson et al., 1990Go; Hendrickson, 1991Go, 1995Go). Recombinant proteins containing selenomethionine residues can be prepared by adding selenomethionine to the culture. It has been reported (Gassner et al., 1999Go) that the substitution of selenomethionine for methionine residues enhanced the stability of T4 lysozyme and the protein had a similar structure to the intact protein. However, in general, methionine residues in proteins have been observed with low frequency [2.4% (MaCaldon and Argos, 1988Go)].

From a comparison of many closely related protein structures, the `safe' residue substitutions that do not disturb the protein structure were shown (Bordo and Argos, 1991Go) (Figure 1Go). Leucine was shown to be the only candidate for `safe' residue substitution of methionine and has a similar hydrophobicity to methionine [difference ~0.5 kcal/mol (Finney et al., 1980Go; Guy, 1985Go)]. Experimental data presented in a previous report (Gassner et al., 1996Go) showed that the loss of stability in the mutant T4 lysozyme where leucine was mutated to methionine was from –0.4 to –0.8 kcal/mol (for only one mutation was it –1.9 kcal/mol). However, all leucine residues cannot always be substituted for methionine residues and they may not be suitably located where a selenomethionine residue should be introduced. Therefore, another substituent of methionine would be required. As an isoleucine residue has a similar volume to a leucine residue, it should be the next candidate for the choice of a substituent for methionine. However, isoleucine was not considered to be a `safe' residue for substitutions of methionine (Bordo and Argos, 1991Go). The change in hydrophobicity from isoleucine to methionine was larger than that from leucine to methionine by about 0.5 kcal/mol (Finney et al., 1980Go; Guy, 1985Go). The instabilities of two mutants of T4 lysozyme where isoleucine at the hydrophobic core was mutated to methionine were 1.5 and 1.6 kcal/mol (Gassner et al., 1996Go). Hence it is desirable to examine the possibility of substitution of methionine for isoleucine. The secure substitution of methionine for isoleucine in a protein molecule means being able to introduce selenomethionine into the position where isoleucine is located. Therefore, it is useful to examine whether isoleucine in protein structures is replaced with methionine for the development of a method for the determination of protein structure using X-ray crystallography. So far, substitutions of methionine for isoleucine have been reported in several proteins (Leahy et al., 1994Go; Skinner et al., 1994Go; Gassner and Matthews, 1999Go). However, these substitutions of methionine for isoleucine were conducted only in hydrophobic cores or in completely buried positions, and not systematically.



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Fig. 1. This illustration of `safe' residue substitutions is partially taken from Figure 2Go of Bordo and Argos (1991). The large circle shows that residues roughly equivalent are grouped together in a subset, which generally correlate with side chain physicochemical properties. Statistically preferred substitutions observed in buried residues (dashed segments) and exposed residues (solid segments).

 
Hen lysozyme has six isoleucine residues at positions 55, 58, 78, 88, 98 and 124 (Figure 2Go). These are divided into three categories concerning location, i.e. the pocket in the protein surface (residues 78 and 124), {alpha}-helix in the protein (residues 88 and 98) and ß-sheet in the protein (residues 55 and 58). Each isoleucine residue was buried ~81% (residue 78), ~74% (residue 124), ~95% (residues 88 and 98) and completely (residues 55 and 58). Since the residues of the same category were similar in not only secondary structure but also solvent-accessible surface area, the study of substitution may be sufficient to analyze one residue each in a category.



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Fig. 2. Ribbon drawing showing the location of isoleucine residues in hen lysozyme. Isoleucine side chains are shown as ball-and-stick models.

 
In this work, we mutated three isoleucine residues at positions 58 (protein interior and ß-sheet), 78 (protein surface pocket and loop region) and 98 (protein interior and {alpha}-helix) (Figure 2Go) to methionine and analyzed the stabilities and structures of the resulting mutant lysozymes.


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

Restriction enzymes, T4 polynucleotide kinase and DNA polymerase I (Klenow fragment) were purchased from either Takara Shuzo (Kyoto, Japan) or New England Biolabs (Beverly, MA). Micrococcus luteus and DNA sequencing kits (Sequenase) were purchased from Sigma and Amersham-Pharmacia Japan (Tokyo, Japan), respectively. CM-Toyopearl 650M, a cation-exchange resin for the purification of secreted hen lysozymes, was obtained from Tosoh (Tokyo, Japan).

Mutant lysozymes

Mutation of the lysozyme gene was carried out by site-directed mutagenesis using the method of Kunkel et al. (Kunkel et al., 1987Go), as described previously (Hashimoto et al., 1996Go). The DNA oligonucleotides used as the 5'- and 3'-mutagenic primers (about 20 bases long) were designed such that the codon for Ile58, -78 or -98 was changed from ATC to ATG for Met. For the construction of the single mutant proteins at three isoleucine residues (58, 78 and 98), the wild-type lysozyme (WT) gene in the M13 phage pKP1700 was used as a template. The mutant lysozymes where Ile58, -78 and -98 were mutated to Met are abbreviated to I58M, I78M and I98M, respectively.

Purification and identification of lysozyme secreted by yeast

Each transformant of Saccharomyces cerevisiae AH22 was cultivated at 30°C for 125 h for expression and secretion of the respective mutant lysozyme (Hashimoto et al., 1996Go). Purification (ion-exchange chromatography) and identification (peptide mapping, amino acid sequencing and amino acid composition) of the lysozymes were carried out as reported previously (Ueda et al., 1993Go).

Unfolding equilibrium

The unfolding equilibrium of lysozymes by guanidine hydrochloride (Gdn.HCl) was determined at pH 5.5 and 35°C by measuring the fluorescence at 360 nm (excitation at 280 nm). The protein concentration was 0.9x10–7 M. The buffer used was 0.1 M sodium acetate adjusted to pH 5.5 with HCl. It has been found experimentally that the free energy of unfolding of proteins in the presence of Gdn.HCl is linearly related to the concentration of denaturant (Pace, 1986Go). Details of the analysis were as described (Inoue et al., 1992Go) and the average m value of WT and isoleucine to methionine (IM) mutants was employed.

X-ray analysis

Crystallization was carried out using a hanging drop vapor diffusion technique at pH 4.7. Intensity data collection for WT and IM mutants was carried out with an R-AXIS IIC automated oscillation camera system (Rigaku), equipped with an imaging plate detector, on a Cu rotating-anode generator operated at 40 keV and 120 mA at room temperature. Refinement of the structure was carried out using the programs TURBO-FRODO (Roussel et al., 1990Go) and X-PLOR (Brunger, 1992Go) installed on an SGI Indigo2. The crystallographic data for WT and IM mutants are shown in Table IGo. Each sample was measured at a resolution in the range 1.63–1.91 Å, R-merge was <9% and data completeness was >84%. The refinements of the mutant lysozymes were carried out as described previously (Ohmura et al., 1997Go). Coordinates for WT have been deposited in the Protein Data Bank (Brookhaven National Laboratory) (accession number 1RFP) and coordinates for all IM mutants (I58M, I78M and I98M) have also been deposited in the Protein Data Bank.


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Table I. Crystallographic data collection and refinement statistics
 

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 Materials and methods
 Results
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Mutant stabilities

We designed three mutants in which Ile is mutated to Met in lysozyme (IM mutant). Position 58 is located in an anti-parallel ß-sheet and buried in the interior of the lysozyme molecule. Position 78 is located on the surface in lysozyme molecule but partially covered by the nearby residues. Position 98 is located in an {alpha}-helix and buried in the interior of the lysozyme molecule. Stabilities of all the IM mutants were measured by a Gdn.HCl denaturation experiment. I58M was less stable than WT by about 1.2 kcal/mol (Table IIGo). Both I78M and I98M were less stable than WT by 0.9 kcal/mol (Table IIGo).


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Table II. Thermodynamic parameters of lysozymes for the denaturation against Gdn.HCl at pH 5.5
 
Crystal structures of IM mutants

After the refinement of the crystal structures, R-factors of all mutants fell to ~17%. The overall and local structures of all IM mutants were similar to those of WT (Figures 3 and 4GoGo). The side chains of all substituted methionine residues were placed mimicking the C{gamma}1–C{delta}1 chain of isoleucine residue in WT (Figure 4Go). The dihedral angle and average B-factor of the side chain of the mutated residue are shown in Table IIIGo. There were no unfavorable contacts in these structures (Figure 5Go). The solvent-accessible surface areas at the mutated residue in IM mutants (I58M, 3; I78M, 36; and I98M, 10 Å2) were almost the same as those of WT (I58, 1; I78, 34; and I98, 8 Å2).



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Fig. 3. Plots of the root-mean-square deviation of main chain for WT versus residue number for I58M (thin line), I78M (dashed line) and I98M (thick line).

 




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Fig. 4. Comparisons of each mutated region in the refined structures of the mutant (white circles) and WT structure (gray circles). The alignments are based on the least-squares superposition of the main-chain atoms. (A) I58M versus WT, (B) I78M versus WT and (C) I98M versus WT.

 

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Table III. Dihedral angles and average B-factors of side chain for the mutated residues
 




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Fig. 5. Stereo-views of space-filling models of each mutated region in the refined structures of the mutant lysozymes. They show the substituted residue. (A) I58M (B) I78M and (C) I98M.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
All mutant lysozymes became slightly unstable by about 1.0 kcal/mol. The value of the instability was in reasonable agreement with the difference in the solvent transfer free energy ({Delta}{Delta}Gtr) between methionine and isoleucine (Finney et al., 1980Go; Guy, 1985Go). Therefore, the instability of all IM mutant lysozymes may be explained by the change in the hydrophobicity from isoleucine to methionine. This was consistent with the results that there were no unfavorable contacts based on X-ray structures of all IM mutant lysozymes. Thus, it was found that the substitution of methionine for isoleucine in various secondary structure regions evoked a similar effect on the protein stability only of the hydrophobicity change. The instability of IM mutant in T4 lysozyme was also in good agreement with Finney's {Delta}{Delta}Gtr from isoleucine to methionine [about 1.5 kcal/mol (Gassner et al., 1996Go)]. Therefore, this result also supports the idea that the instability of an IM mutant in a protein may be determined by the effect of the change of hydrophobicity from isoleucine to methionine.

The side chain atoms (Cß, C{gamma} and S{delta}) in methionine of the respective mutant lysozymes were located at overlapped positions with the respective side chain atoms (Cß, C{gamma}1 and C{delta}1) of isoleucine in WT (Figure 4Go). As a result, there were few changes in local structures and in the solvent-accessible surface areas in mutant lysozymes. The crystal structure of gene V protein where Ile47 is mutated to Met has been solved at 1.8 Å resolution (Skinner et al., 1994Go). The conformation of methionine in this mutant protein was also located at an overlapped position in side chain atoms of isoleucine in the intact protein. This result was consistent with the present results. Therefore, these coincidences of the side chain conformation in a protein where isoleucine residues are mutated to methionine residues indicated that the side chain conformation of methionine in a mutant protein may mimic that of isoleucine in an intact protein.

The dihedral angles of the side chain of the mutated residues did not variy except for {chi}1 of I78M (Table IIIGo). The change of conformation energy calculated for {chi}1 of residue 78 was small. Therefore, it would hardly influence the protein stability. The average B-factors of the side chains of the mutated residues were slightly larger than that of WT (Table IIIGo). The increased B-factor of the side chain may reflect the increased entropy and/or the decreased enthalpy of the mutated residue. Funahashi et al. reported on the protein stability of IM mutant at positions 56 and 59 of human lysozyme (Funahashi et al., 1999Go). The I59M in human lysozyme corresponds to I58M in hen lysozyme. Decreased enthalpies were observed in IM mutants of human lysozyme. The same explanation would be applicable to IM mutant of hen lysozyme, although the result for human lysozyme could not be applied simply to that for hen lysozyme. X-ray crystallographic data show that the errors in the data for mutant lysozymes were slightly larger than that in WT (Table IGo). The largest B-factor was seen in mutated residue 78. Residue 78 was buried 81% and the B-factor seen here was comparable to those of the residues having similar accessibility. As a result, it could be considered that the observed increase in B-factors in the mutant residues deserves no special attention. Hence we can conclude that the substitution of methionine for isoleucine did not cause substantial structural changes.

Other workers also reported the security of the substitution of methionine for isoleucine (Leahy et al., 1994Go; Skinner et al., 1994Go; Gasnner and Matthews, 1999,). Thus, in general, the substitution of methionine for isoleucine would be considered as a `safe' residue substitution in a protein. Although Gasnner and Matthews mentioned that the second-best substitution is Phe -> Met rather than Ile -> Met, we thought that the second-best substitution would be Ile -> Met in terms of hydrophobicity, side chain volume and Dayhoff mutation probability.

In conclusion, it has been shown that the substitution of methionine for isoleucine in a protein could be performed without any significant change of the overall and local structure. Thus, isoleucine would be the second candidate after leucine as the substituent of methionine. Hence this substituent could be considered as a `safe' residue substitution but the reverse substitution remains unknown. Our present findings provide valuable information on point mutations of amino acid residues in a protein and may contribute to the determination of novel protein structures with the mutation from isoleucine to selenomethionine by using the multiple-wavelength anomalous dispersion method.


    Notes
 
1 To whom correspondence should be addressed. E-mail: imoto{at}phar.kyushu-u.ac.jp Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Received October 12, 2000; revised February 21, 2001; accepted March 5, 2001.