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X-ray crystallography and structural stability of digestive lysozyme from cow stomach Yasuhiro Nonaka1, Daisuke Akieda1, Tomoyasu Aizawa1, Nobuhisa Watanabe1,2, Masakatsu Kamiya3, Yasuhiro Kumaki1, Mineyuki Mizuguchi4, Takashi Kikukawa1, Makoto Demura3 and Keiichi Kawano1 1 2 3 4 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan Department of Biotechnology and Biomaterial Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japan Division of Molecular Life Science, Graduate School of Life Science, Hokkaido University, Sapporo, Japan Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan Keywords lysozyme; molecular evolution; protease resistance; structural stability; X-ray crystallography Correspondence K. Kawano, Graduate School of Science, Hokkaido University, North 10, West 8, Kita-ku, Sapporo, Hokkaido 060 0810, Japan Fax: +81 11 706 2770 Tel: +81 11 706 2770 E-mail: kawano@mail.sci.hokudai.ac.jp (Received 12 November 2008, revised 22 January 2009, accepted 4 February 2009) In ruminants, some leaf-eating animals, and some insects, defensive lysozymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resistant to protease and to have optimal activity at acidic pH. The structural basis of the adaptation providing persistence of lytic activity under severe gastric conditions remains unclear. In this investigation, we obtained the crystallographic structure of recombinant bovine stomach lysozyme 2 (BSL2). Our denaturant and thermal unfolding experiments revealed that BSL2 has high conformational stability at acidic pH. The high stability in acidic solution could be related to pepsin resistance, which has been previously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could provide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin. doi:10.1111/j.1742-4658.2009.06948.x C-type lysozyme (EC 3.2.1.17), represented by hen egg-white lysozyme (HEWL), is one of the most well-known enzymes. It has been found in various vertebrates, arthropods, and some other metazoa. It catalyzes the hydrolysis of the b-1,4-glycoside linkage between N-acetylglucosamine and N-acetylmuramic acid of peptidoglycan, and thus breaks the bacterial cell wall [1]. Most c-type lysozymes reported thus far are considered to play a role in defense against bacterial infection. It was proposed that the bacteriolytic activity of lysozymes is also used for digestion in some species. In artiodactyl ruminants, which feed on plants, the foregut chamber has evolved to digest cellulose efficiently [2–4]. They recruit bacteria that ferment cellulose in the foregut. The bacteria are broken down by lysozyme in the true stomach, and the digested component is then absorbed in the intestine. The acquisition of digestive lysozyme is well known as a case of convergent evolution [4]. In addition to artiodactyla, many other animals, such as a folivorous monkey (colobus) and a bird (hoatzin), as well as the house fly, are known to have digestive c-type lysozymes [5–7]. Those folivorous animals obtain nourishment from plant material in a similar manner to artiodactyla. House fly larvae feed on bacteria growing in decomposing material, and digest the bacteria with lysozyme. According to phylogenetic analyses, each phylogenetic group has independently adapted its defensive lysozyme for digestion [7,8]. Interestingly, common Abbreviations BSL2, bovine stomach lysozyme 2; DSC, differential scanning calorimetry; HEWL, hen egg-white lysozyme. 2192 FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS Y. Nonaka et al. Structure and stability of bovine stomach lysozyme properties, e.g. low optimal pH and resistance to protease, are shared by digestive lysozymes from different organisms [6,8–10]. Furthermore, ruminant and colobus lysozymes share similarities in amino acid sequence, and this is unlikely to have occurred by random drift, suggesting convergent (or parallel) amino acid replacements [7]. These functional and structural similarities could have resulted from adaptation to severe gastric conditions. However, the molecular bases for such adaptations remain to be investigated. Recently, the crystal structure of house fly digestive lysozyme was solved, explaining the mechanism underlying the acidic pH optimum [11]. The pKa values of the catalytic residues are lowered by neighboring residues, resulting in the acidic pH optimum. No experimental three-dimensional structure of vertebrate digestive lysozyme has been reported thus far. It would be useful to understand the structural bases for the adaptation by comparing this lysozyme with house fly digestive and other nondigestive lysozymes. In this study, we obtained recombinant bovine stomach lysozyme 2 (BSL2), the most highly expressed lysozyme in the cow stomach. X-ray crystallography and some other experiments were performed to determine how this lysozyme has acquired the properties mentioned above. We also discuss the significance of the probable convergent amino acid replacements. Results X-ray crystallography of BSL2 The crystal structure of BSL2 is shown in Fig. 1A, and the data collection, processing and refinement statistics are summarized in Table 1. BSL2 was crystallized in the space group P212121. The structure was refined at 1.5 Å to an R-factor of 17.8% and an R-free of Table 1. Data collection, processing and refinement statistics. Data collection Space group Cell constants (Å) a b c Resolution (Å) No. observations I ⁄ r(I) No. unique reflections Rmerge Completeness (%) Multiplicity Refinement data Resolution (Å) No. reflections R-factor Rfree Rmsd from ideal values Bond lengths (Å) Bond angles () a P212121 31.257 56.065 64.050 50.00–1.50 (1.55–1.50)a 126 692 28.085 (17.272) 17833 (1662) 0.046 (0.088) 95.0 (90.7) 7.1 (7.1) 17.94–1.50 16 849 0.178 0.221 0.009 1.261 Values in parentheses are for the last resolution shell. 22.1%. The average B-value for all protein atoms is 10.17 Å2, and that for all main chain atoms is 9.25 Å2. The electron density map was sufficiently clear to build a molecular model, and most of the side chain conformations were determined unequivocally, although some residues showed multiple conformers. This lysozyme is composed of an a-domain and a b-domain, both of which are common in the previously reported structures for other c-type lysozymes. The a-domain is composed of four a-helices (A–D), and the b-domain is composed of a large loop and a three-strand antiparallel b-sheet. Figure 1B is a superimposition of the main chain conformations of BSL2, human lysozyme, HEWL, and house fly midgut A B Fig. 1. (A) Ribbon model of BSL2 (Protein Data Bank ID: 2Z2F) in which a-helices are sequentially labeled from A to D. The structure is shown in rainbow colors from the N-terminus to the C-terminus. The figure was produced using MOLFEAT (FiatLux, Tokyo, Japan). (B) Superimposition of the Ca conformation of BSL2 (red), human lysozyme (green, 1JSF), HEWL (blue, 1DPX), and house fly midgut lysozyme (yellow, A chain of 2FBD). The broken-line circle represents the loop region following the C-helix. The figure was produced using MOLMOL [50]. FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 2193 Structure and stability of bovine stomach lysozyme Y. Nonaka et al. lysozyme. The rmsd between BSL2 and human lysozyme, calculated using the backbone atoms in the a-helices, is 0.38 Å, that between BSL2 and HEWL is 0.35 Å, and that between BSL2 and house fly lysozyme is 0.79 Å. The backbone structure of BSL2 is closer to that of the vertebrate nondigestive lysozyme than to that of insect digestive lysozyme. A B C pH dependence of the lytic activity of BSL2 The digestive lysozymes reported thus far tend to have a pH optimum at acidic pH, whereas nondigestive lysozymes have a broad optimum at neutral pH [8,9]. The relative lytic activities of recombinant BSL2 and commercial HEWL at pH 4–7 are shown in Fig. 2. The pH optimum of BSL2 was about 5, whereas that of HEWL occurred at pH values higher than 6. BSL2 exhibited less activity than HEWL, even at the optimal pH of BSL2. At pH 7, BSL2 showed almost no lytic activity. Structural stability of BSL2 in acidic conditions Digestive lysozymes need protease resistance to maintain their lytic activity in the stomach. As shown in Fig. 3, BSL2 is more resistant than HEWL to pepsin. Pepsin readily digested HEWL in acidic conditions with physiological ionic strength (150 mm NaCl), whereas BSL2 remained intact after 4 h. This result corresponded to that for natural BSL2 from bovine stomach, based on residual activity [9]. Fig. 2. Bacteriolytic activities of BSL2 (gray bars) and HEWL (white bars) at different pH values, ionic strength 0.1, and 25 C. The relative activities are expressed by taking the activity of HEWL at pH 7.0 as 1.0. 2194 Fig. 3. SDS ⁄ PAGE of pepsin-treated BSL2 and HEWL with (A) 0 mM NaCl (B) 150 mM NaCl, and (C) 500 mM NaCl. Aliquots of the solution were sampled at intervals of 1 h. M is the marker lane. In one report, protease resistance was correlated with protein thermostability [12]. To evaluate the structural stability of BSL2, denaturant-induced unfolding and thermal unfolding were monitored. Figure 4 shows the guanidinium hydrochloride-unfolding curves of BSL2 and HEWL, as determined by CD ellipticity at 222 nm, indicating the disruption of the native structure. The parameters derived from these unfolding curves are shown in Table 2. At pH 6.0, BSL2 and HEWL were similar in their midpoints (Cm), Gibbs free energies without denaturant (DGw), and m values indicative of cooperativity. At pH 2.0, in contrast, BSL2 unfolded at a higher concentration of guanidinium hydrochloride than HEWL. The Gibbs free energy of BSL2 at low pH was much greater than that of HEWL, indicating the high conformational stability of BSL2. The transition temperatures (Tm) and unfolding enthalpy values (DHu) at pH 2.0, obtained by thermal unfolding experiments using differential scanning calorimetry (DSC), are also summarized in Table 2. BSL2 unfolded at a higher temperature and had a greater DHu value, also indicating greater structural stability. Hydrogen exchange properties were monitored by 1D 1H-NMR at pH 1.9, to compare the conformational flexibilities of BSL2 and HEWL (Fig. 5). Generally, there are few or no peaks around 10 p.p.m., except for the peaks of tryptophan indole hydrogen atoms. Both BSL2 and HEWL have six tryptophan residues, and five peaks appear around 10 p.p.m. for both proteins. In the spectra of HEWL, most of the indole hydrogen peaks diminished rapidly within 30–60 min, and only the peak at 10.3 p.p.m. remained after a 120 min exchange. In the spectra of BSL2, three peaks were observed after the 30 min exchange, and decreased gradually. In particular, the peak of Trp64 in BSL2 diminishes more slowly than that of the corresponding residue, Trp63, in HEWL. The tryptophan residues whose peaks diminished rather slowly could exist in rigid and unexposed regions. FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS W34 Normalized intensity W64 A W108 Structure and stability of bovine stomach lysozyme W111 W63 Y. Nonaka et al. 10.6 10.4 p.p.m. 10.2 10.0 9.8 Normalized intensity W108 10.4 p.p.m. W123 B 10.6 W63 W62 10.8 W111 11.0 Fig. 4. Guanidinium hydrochloride-induced unfolding curves of BSL2 (circles) and HEWL (triangles) monitored by CD at (A) pH 2.0 and (B) pH 6.0. The apparent fractions of unfolding protein, fapp, were plotted against the concentration of guanidinium hydrochloride. The lines are the transition curves estimated by the nonlinear least squares method. 11.0 10.8 10.2 10.0 9.8 Fig. 5. 1D 1H-NMR spectra of (A) BSL2 and (B) HEWL in 95% H2O ⁄ 5% D2O (thick lines) and after 30, 60 or 120 min of hydrogen–deuterium exchange in 100% D2O (thin lines). The spectra were acquired at pH 1.9. Table 2. Thermodynamic parameters for guanidinium hydrochloride-induced and thermal unfolding. pH 2.0 BSL2 pH 6.0 HEWL Guanidinium hydrochloride-induced unfolding Cm (M) 3.07 2.17 DGw (kJÆmol)1) 32.9 17.3 m (kJÆmol)1 M) 10.7 7.97 Thermal unfolding Tm (K) 333.8 326.6 406.4 386.4 DH (kJÆmol)1)a a BSL2 HEWL 4.17 53.4 12.8 4.16 41.9 10.1 The unfolding enthalpies at transition temperature Tm. Discussion Although BSL2 has an acidic optimal pH, the relative activity level is lower than or comparable to that of HEWL, even at acidic pH (Fig. 2). BSL2, like many acidophilic proteins [13–15], possesses a greater number of acidic residues than nondigestive lysozymes (Table 3). An increase in acidic residues would result in low lytic activity, because the electrostatic attraction between the lysozyme and the negatively charged bacterial membrane becomes weaker, especially at neutral pH. BSL isozymes are considered to function below pH 6 in nature [9]. It is likely that BSL2 has lost lytic activity at neutral pH and retains it below pH 6. In the case of house fly digestive lysozyme, the crystallographic analysis and catalytic activity experiments indicated that the catalytic residues have lower pKa values than those of HEWL, and thus the optimal pH is shifted to the acidic range [11]. Using the crystallographic structures, we calculated the pKa values of the FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 2195 Structure and stability of bovine stomach lysozyme Y. Nonaka et al. Table 3. Comparison of structural parameters among lysozymes. No. of residues No. of charged residues Negative Positive No. of salt bridges No. of hydrogen bonds Hydrogen bonds ⁄ residue BSL2 HEWL Human House fly 129 129 130 122 15 18 4 125 0.97 9 18 3 122 0.95 11 20 5 125 0.96 9 12 3 109 0.89 catalytic residues Glu35 and Asp52 (numbering for HEWL), for BSL2 and other lysozymes, with propka 2.0 [16]. The predicted pKa values were 6.15 and 4.27 for BSL2, 5.93 and 4.20 for HEWL, and 4.89 and 3.84 for house fly lysozyme. Although these values do not agree completely with the experimental results [11], the acidic shifts of the pKa values for house fly lysozyme are well predicted. The calculated pKa values for BSL2 are not reduced as compared to those for HEWL. Glu35 in BSL2 is surrounded by hydrophobic residues, as it is in HEWL, and this results in the high pKa, whereas the polarity of Thr110 reduces the pKa for house fly lysozyme. In the case of Asp52, the pKa is modulated by the hydrogen bond network. There are hydrogen bonds formed by Asp52, Asn46 and Asp48 in HEWL. House fly lysozyme has an asparagine at position 48, and the absence of the negative charge should reduce the pKa of Asp52 as compared to HEWL [11]. Asn46 in BSL2 is distant from Asp52, and the absence of this hydrogen bond network would reduce the pKa. However, Asp52 in BSL2 is more exposed to solvent than that in HEWL, and this raises the pKa. As a result, the calculated pKa values for BSL2 were comparable to those for HEWL. The result suggests that the catalytic activity of BSL2 is not adapted to acidic conditions, unlike the case with house fly lysozyme. BSL2 and other vertebrate digestive lysozymes have been reported to be resistant to pepsin digestion, as is also shown in Fig. 3. The efficiency of peptide bond fission by protease reflects the conformational flexibility of the polypeptide substrate [12,17,18]. The correlation between structural rigidity and stability has been reported for many proteins [19–22]. The high conformational stability of BSL2 as compared to HEWL (Table 2) suggests greater structural rigidity. The higher rigidity was also suggested by the hydrogen exchange experiment (Fig. 5). Trp64 in BSL2 is protected, whereas Trp63 in HEWL is not. This residue exists in the b-domain, and is oriented to the interface between the two domains. Therefore, the interface of BSL2 is less susceptible to unfolding than that of 2196 HEWL. These results support the notion that conformational rigidity protects BSL2 from pepsin digestion. Because the house fly lysozyme is resistant to cathepsin D, a protease from the house fly midgut [5], the house fly midgut lysozyme would have structural stability and rigidity similar to that of BSL2. As observed for thermophilic enzymes, an increase in conformational rigidity often leads to a reduction in enzymatic activity [22–24]. The lower lytic activity of BSL2 (Fig. 2) may also be caused by the increased rigidity, and not only by the increased negative charge. The numbers of positive and negative charges differ among these lysozymes (Table 3). The surfaces of HEWL and human lysozyme are predominantly positively charged. A lysozyme covered with positively charged surfaces will have a loose structure, because electrostatic repulsion significantly increases on the molecular surface. BSL2 has a negatively charged b-domain and a positively charged a-domain. The electrostatic repulsion on the surface will be weaker, and this could contribute to the higher stability. There are fewer charged residues on the surface of the house fly lysozyme, and the electrostatic repulsion will be smaller. The house fly lysozyme may have achieved structural stability by decreasing the positively charged residues. The increase in acidic residues is also expected to result in an increase in the number of salt bridges. The numbers of the salt bridges in BSL2 and HEWL, however, are comparable (Table 3). It is noteworthy that BSL2 contains a complex salt bridge (Glu83–Lys91– Glu86) that is absent in the three other lysozymes. A triangular salt bridge formed by two acidic residues and one basic residue can be more strong than the sum of simple salt bridges [25–27]. The loop located between Glu83 and Lys91 connects the b-domain and the a-domain. In the case of calcium-binding lysozyme, calcium binding at this loop stabilizes the native structure [28,29]. By analogy, the electrostatic interaction at this loop is considered to contribute to the overall structural stability. The overall structures of these lysozymes are very similar (Fig. 1B), and the numbers of hydrogen bonds are comparable (Table 3). A marked difference is observed in the region from the C-terminus of the C-helix to the following loop, residues 100–103 in HEWL (Fig. 1B). The C-helices of human lysozyme and HEWL are terminated at residue 101 followed by proline or glycine, which can destabilize the a-helix [30]. BSL2 and house fly lysozyme lack this proline or glycine residue, and thus the C-helices are longer and the following loops are shorter than those of HEWL and human lysozyme. This would prevent pepsin FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS Y. Nonaka et al. digestion, because there are proteolytic sites for pepsin in this loop for HEWL and human lysozyme [18,31]. The amino acid replacements at positions 14, 21, 50, 75 and 87 were considered to be significant for the adaptation of digestive lysozyme, on the basis of the analyses using vertebrate digestive and nondigestive lysozyme sequences [7,32]. No remarkable difference, such as the alteration of hydrogen bonds, is found at these positions between BSL2 and human lysozyme, except at residue 21. The side chain of Lys21 in BSL2 forms hydrogen bonds with the side chains of Tyr20 and Ser101, whereas the side chain of Arg21 in human lysozyme hydrogen-bonds to the backbone carbonyl oxygens of Val100 and Asp102. As discussed above, the region that includes residues 100–102 could be associated with resistance to pepsin. The replacement of residue 21 could also be an adaptation to stabilize this region. Experimental procedures Expression and purification of BSL2 In an Escherichia coli expression system, removal of an extra methionine residue at the N-terminus does not take place in the case of lysozyme [33]. We obtained recombinant BSL2 with a perfect sequence using the methylotrophic yeast Pichia pastoris, basically as described by Digan et al. [34]. The cDNA was ligated to the expression vector pPIC3 (Invitrogen, Carlsbad, CA, USA). To secrete BSL2 into the culture, we incorporated the native signal sequence of BSL2. The plasmid was linearized by SalI, and transformed into P. pastoris GS115 by electroporation. Genotypic selection and phenotypic screening were performed on a minimal dextrose plate (1.34% yeast nitrogen base, 4 · 10)5% biotin, 1% dextrose, and 1.5% agar) and on a minimal methanol lysoplate (1.34% yeast nitrogen base, 4 · 10)5% biotin, 0.061% Micrococcus lysodeikticus, and 1.5% agar, in 10 mm potassium phosphate buffer, pH 5.0), as previously reported, except for pH and buffer concentration [35]. Colonies on a minimal dextrose plate were inoculated onto a minimal methanol lysoplate, and 200 lL of methanol was spread on the plate cover and incubated at 30 C for about 1–3 days. The radius of the translucent plaque around the colony was measured as an indicator of the colony’s lysozyme expression level. P. pastoris for BSL2 expression was cultivated using a jar fermenter with high-density fermentation [36–38]. To avoid proteolysis, we recovered the culture after induction for 48 h. To purify recombinant lysozyme using cation exchange chromatography, the supernatant of the culture was diluted so that the electrical conductivity was decreased to < 5 mSÆcm)1. The diluted supernatant was filtered Structure and stability of bovine stomach lysozyme through a nitrocellulose membrane. The supernatant was loaded onto an SP-Sepharose Fast Flow column (300 mL) (GE Healthcare, Piscataway, NJ, USA) equilibrated with 50 mm sodium acetate buffer (pH 4.8), and the adsorbed proteins were eluted with 50 mm sodium acetate buffer with 1 m NaCl (pH 4.8). The elution was monitored by absorbance at 280 nm. The sample solution was dialyzed with 50 mm sodium acetate buffer (pH 4.8) to decrease electrical conductivity. After dialysis, the sample was loaded onto an SP-Sepharose Fast Flow column equilibrated with 50 mm sodium acetate buffer (pH 4.8), and eluted with a salt linear gradient of 50 mm sodium acetate buffer with 1 m NaCl (pH 4.8). The main peak fraction was dialyzed with 20 mm NH4HCO3 and freeze-dried. Assay of lytic activity The lytic activities of BSL2 and HEWL against M. lysodeikticus were estimated using the turbidimetric method [39]. Lyophilized M. lysodeikticus was purchased from Sigma-Aldrich (St Louis, MO, USA). Suspensions of M. lysodeikticus were prepared in sodium acetate (for pH 4 and 5) and sodium phosphate (for pH 6 and 7) buffer. The ionic strength of each buffer was adjusted to 0.1 [40]. Lysozyme solution and M. lysodeikticus suspension were mixed, and the decrease in absorbance was monitored at 540 nm with a thermostatically controlled cell holder at 25 C. The relative activity was calculated from the speed of the absorbance decrement. Pepsin digestion Pepsin was obtained from Sigma-Aldrich. HEWL was obtained from Seikagaku Corp. (Tokyo, Japan). Lysozymes were dissolved in 10 mm HCl (pH 2), and the final protein concentration was 0.5 mgÆmL)1. The digestion experiment was carried out in the presence of pepsin at 37 C. The aliquots were sampled at intervals of 1 h and then frozen until electrophoresis. X-ray crystallography A crystal of BSL2 was obtained by the vapor diffusion (sitting drop) method, using 0.1 m sodium Hepes buffer at pH 7.5, containing 0.2 m NaCl and 30% 2-methyl-2,4-pentanediol. The space group of the crystal was P212121, with cell dimensions a = 31.257 Å, b = 56.065 Å, and c = 64.050 Å. There is one monomeric molecule in an asymmetric unit. The X-ray diffraction data of BSL2 were collected from a single crystal at 93 K, using a MicroMAX-007 generator (Rigaku, Tokyo, Japan) and an R-AXIS IV++ detector (Rigaku). The reflections were processed with the program hkl-2000 [41]. The I ⁄ r(I) in the last resolution shell (1.55–1.50) was 17.272. The resolution was limited by the FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 2197 Structure and stability of bovine stomach lysozyme Y. Nonaka et al. acceptance of the detector. The limit at the edge of the detector using an 80 mm crystal-to-film distance is approximately 1.5 Å resolution. The structure was solved by the molecular replacement method, using the program molrep [42] packaged in ccp4 [43]. The structure of recombinant human lysozyme (Protein Data Bank code: 1LZ1) [44] was used as the search model. The structure was refined using the program refmac5 [45] in the ccp4 suite, and was visually inspected using coot [46]. Water molecules were found by the functions in refmac5 and coot, and were checked visually using coot. A sodium ion was added to the model as judged by the electron density, coordination number, and interatomic distance. The structure was deposited in the Protein Data Bank under the code 2Z2F. Analysis of structural features A salt bridge in Table 3 was defined as a negative residue and a positive residue with an interatomic distance of < 4.0 Å. The hydrogen bonds were detected using the what if web interface with the following criteria: maximal distances of 3.5 Å for donor–acceptor and 2.5 Å for hydrogen–acceptor, and minimal angles of 60 for donor– hydrogen–acceptor and 90 for hydrogen–acceptor–X. Water-mediated hydrogen bonds were not included. CD CD at 222 nm was measured with a Jasco J-725 spectropolarimeter (Japan Spectroscopic, Tokyo, Japan), using optical cells with path length of 1 mm. The guanidinium hydrochloride-induced unfolding experiment was carried out at 298 K using 50 mm KCl ⁄ HCl buffer at pH 2.0, and 50 mm sodium phosphate buffer at pH 6.0. The concentration of guanidinium hydrochloride was determined by the difference between the refractive indices of guanidinium hydrochloride solution and guanidinium hydrochloride-free solution. The protein concentration was 8–10 lm. The unfolding curves were fitted to the following equation: DG = – RTlnK = DGw – mC, where DG and DGw are the Gibbs free energy with denaturant and that without denaturant respectively, and R, T, K, m and C are the gas constant, absolute temperature, equilibrium constant, cooperativity index, and denaturant concentration, respectively. DSC DSC was carried out using VP-DSC (MicroCal, Northampton, MA, USA), at a scan rate of 1.0 KÆmin)1. Sample solution was prepared with reference buffer 50 mm glycineHCl at pH 2.0. To extend the temperature range, all DSC measurements were performed under a pressure of 2.0 atm. The protein concentration and pH were confirmed after the 2198 scan. The DSC curves were analyzed to obtain the transition temperatures (Tm) and unfolding enthalpies (DHu) [47]. Hydrogen–deuterium exchange experiment Hydrogen–deuterium exchange was measured by 1D 1HNMR performed on a Bruker 500 MHz instrument (Bruker BioSpin, Rheinstetten, Germany), with a cryogenic probe and a JEOL ECA-600 instrument (JEOL, Tokyo, Japan). The exchange was initiated by dissolving protein that had been lyophilized with pH-adjusted buffer (pH 1.9) in D2O to give a final protein concentration of 0.3 mm in 50 mm sodium phosphate. The sample was incubated at 298 K. A total of 32 scans of each sample were collected at 30 or 60 min intervals. To acquire the spectra before hydrogen exchange, lysozyme solution was subjected to 1H-NMR in the same buffer with 95% H2O ⁄ 5% D2O. The peaks of unexchangeable hydrogens were used to normalize intensity. The peaks of indole hydrogens were assigned on the basis of the BMRB database (bmr1093 and bmr4562 for HEWL and bmr76 for human lysozyme were used), and using proshift [48], a chemical-shift prediction tool. Estimation of protein concentration The protein concentrations were estimated spectrophotometrically by following the extinction coefficients at 280 nm for a 1% solution in a 1 cm cell: E = 28.4 for BSL2, and E = 26.5 for HEWL, estimated using protparam [49]. Acknowledgements This study was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Japan. We thank the staff of the High-Resolution NMR Laboratory, Graduate School of Science, Hokkaido University, for the NMR measurements, Professor I. Tanaka, Graduate School of Life Science, Hokkaido University, for the X-ray crystallography, and Emeritus Professor K. Nitta, Graduate School of Science, Hokkaido University, for helpful advice. References 1 Prager EM & Jolles P (1996) Animal lysozymes c and g: an overview. In Lysozyme: Model Enzymes in Biochemistry and Biology (Jolles P, ed.), pp. 9–31. EXS, Basel. 2 Langer P (1974) Stomach evolution in the artiodactyla. Mammalia 38, 295–314. 3 Janis C (1976) The evolutionary strategy of the equidae and the origins of rumen and cecal digestion. Evolution 30, 757–774. FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS Y. Nonaka et al. 4 Irwin DM (1996) Molecular evolution of ruminant lysozymes. In Lysozyme: Model Enzymes in Biochemistry and Biology (Jolles P, ed.), pp. 347–361. EXS, Basel. 5 Espinoza-Fuentes FP & Terra WR (1987) Physiological adaptations for digesting bacteria – water fluxes and distribution of digestive enzymes in Musca domestica larval midgut. Insect Biochem 17, 809–817. 6 Kornegay JR, Schilling JW & Wilson AC (1994) Molecular adaptation of a leaf-eating bird – stomach lysozyme of the hoatzin. Mol Biol Evol 11, 921–928. 7 Stewart CB, Schilling JW & Wilson AC (1987) Adaptive evolution in the stomach lysozymes of foregut fermenters. Nature 330, 401–404. 8 Prager EM (1996) Adaptive evolution of lysozyme: changes in amino acid sequence, regulation of expression and gene number. In Lysozyme: Model Enzymes in Biochemistry and Biology (Jolles P, ed.), pp. 323–345. EXS, Basel. 9 Dobson DE, Prager EM & Wilson AC (1984) Stomach lysozymes of ruminants. I. Distribution and catalytic properties. J Biol Chem 259, 11607–11616. 10 Stewart CB & Wilson AC (1987) Sequence convergence and functional adaptation of stomach lysozymes from foregut fermenters. Cold Spring Harb Symp Quant Biol 52, 891–899. 11 Cancado FC, Valerio AA, Marana SR & Barbosa JA (2007) The crystal structure of a lysozyme c from housefly Musca domestica, the first structure of a digestive lysozyme. J Struct Biol 160, 83–92. 12 Daniel RM, Cowan DA, Morgan HW & Curran MP (1982) A correlation between protein thermostability and resistance to proteolysis. Biochem J 207, 641–644. 13 Bonisch H, Schmidt CL, Schafer G & Ladenstein R (2002) The structure of the soluble domain of an archaeal rieske iron–sulfur protein at 1.1 A resolution. J Mol Biol 319, 791–805. 14 Sielecki AR, Fedorov AA, Boodhoo A, Andreeva NS & James MNG (1990) Molecular and crystal-structures of monoclinic porcine pepsin refined at 1.8-A resolution. J Mol Biol 214, 143–170. 15 Fushinobu S, Ito K, Konno M, Wakagi T & Matsuzawa H (1998) Crystallographic and mutational analyses of an extremely acidophilic and acid-stable xylanase: biased distribution of acidic residues and importance of Asp37 for catalysis at low pH. Protein Eng 11, 1121– 1128. 16 Li H, Robertson AD & Jensen JH (2005) Very fast empirical prediction and rationalization of protein pKa values. Proteins 61, 704–721. 17 Fontana A, Fassina G, Vita C, Dalzoppo D, Zamai M & Zambonin M (1986) Correlation between sites of limited proteolysis and segmental mobility in thermolysin. Biochemistry 25, 1847–1851. 18 Frare E, Mossuto MF, de Laureto PP, Dumoulin M, Dobson CM & Fontana A (2006) Identification of the Structure and stability of bovine stomach lysozyme 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 core structure of lysozyme amyloid fibrils by proteolysis. J Mol Biol 361, 551–561. Tang KES & Dill KA (1998) Native protein fluctuations: the conformational-motion temperature and the inverse correlation of protein flexibility with protein stability. J Biomol Struct Dyn 16, 397–411. Gershenson A, Schauerte JA, Giver L & Arnold FH (2000) Tryptophan phosphorescence study of enzyme flexibility and unfolding in laboratory-evolved thermostable esterases. Biochemistry 39, 4658–4665. Vihinen M (1987) Relationship of protein flexibility to thermostability. Protein Eng 1, 477–480. Zavodszky P, Kardos J, Svingor A & Petsko GA (1998) Adjustment of conformational flexibility is a key event in the thermal adaptation of proteins. Proc Natl Acad Sci USA 95, 7406–7411. Varley PG & Pain RH (1991) Relation between stability, dynamics and enzyme-activity in 3-phosphoglycerate kinases from yeast and Thermus thermophilus. J Mol Biol 220, 531–538. Wrba A, Schweiger A, Schultes V, Jaenicke R & Zavodszky P (1990) Extremely thermostable d-glyceraldehyde-3-phosphate dehydrogenase from the eubacterium Thermotoga maritima. Biochemistry 29, 7584–7592. Mayne L, Englander SW, Qiu R, Yang JX, Gong YX, Spek EJ & Kallenbach NR (1998) Stabilizing effect of a multiple salt bridge in a prenucleated peptide. J Am Chem Soc 120, 10643–10645. Gvritishvili AG, Gribenko AV & Makhatadze GI (2008) Cooperativity of complex salt bridges. Protein Sci 17, 1285–1290. Horovitz A, Serrano L, Avron B, Bycroft M & Fersht AR (1990) Strength and cooperativity of contributions of surface salt bridges to protein stability. J Mol Biol 216, 1031–1044. Nitta K, Tsuge H & Iwamoto H (1993) Comparative study of the stability of the folding intermediates of the calcium-binding lysozymes. Int J Pept Protein Res 41, 118–123. Kikuchi M, Kawano K & Nitta K (1998) Calcium-binding and structural stability of echidna and canine milk lysozymes. Protein Sci 7, 2150–2155. Fersht A (1999) Structure and Mechanism in Protein Science. W. H. Freeman and Co., New York. Ibrahim HR, Inazaki D, Abdou A, Aoki T & Kim M (2005) Processing of lysozyme at distinct loops by pepsin: a novel action for generating multiple antimicrobial peptide motifs in the newborn stomach. Biochim Biophys Acta Gen Subj 1726, 102–114. Zhang J & Kumar S (1997) Detection of convergent and parallel evolution at the amino acid sequence level. Mol Biol Evol 14, 527–536. Miki T, Yasukochi T, Nagatani H, Furuno M, Orita T, Yamada H, Imoto T & Horiuchi T (1987) Construction FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 2199 Structure and stability of bovine stomach lysozyme 34 35 36 37 38 39 Y. Nonaka et al. of a plasmid vector for the regulatable high-level expression of eukaryotic genes in Escherichia coli – an application to overproduction of chicken lysozyme. Protein Eng 1, 327–332. Digan ME, Lair SV, Brierley RA, Siegel RS, Williams ME, Ellis SB, Kellaris PA, Provow SA, Craig WS, Velicelebi G et al. (1989) Continuous production of a novel lysozyme via secretion from the yeast, Pichia pastoris. Biotechnology 7, 160–164. Koganesawa N, Aizawa T, Masaki K, Matsuura A, Nimori T, Bando H, Kawano K & Nitta K (2001) Construction of an expression system of insect lysozyme lacking thermal stability: the effect of selection of signal sequence on level of expression in the Pichia pastoris expression system. Protein Eng 14, 705–710. Brierley RA, Bussineau C, Kosson R, Melton A & Siegel RS (1990) Fermentation development of recombinant Pichia pastoris expressing the heterologous gene – bovine lysozyme. Ann NY Acad Sci 589, 350–362. Katakura Y, Zhang WH, Zhuang GQ, Omasa T, Kishimoto M, Goto W & Suga KI (1998) Effect of methanol concentration on the production of human beta(2)-glycoprotein I domain V by a recombinant Pichia pastoris: a simple system for the control of methanol concentration using a semiconductor gas sensor. J Ferment Bioeng 86, 482–487. Koganesawa N, Aizawa T, Shimojo H, Miura K, Ohnishi A, Demura M, Hayakawa Y, Nitta K & Kawano K (2002) Expression and purification of a small cytokine growth-blocking peptide from armyworm Pseudaletia separata by an optimized fermentation method using the methylotrophic yeast Pichia pastoris. Protein Expr Purif 25, 416–425. Yoshimura K, Toibana A, Kikuchi K, Kobayashi M, Hayakawa T, Nakahama K, Kikuchi M & Ikehara M (1987) Differences between Saccharomyces cerevisiae and Bacillus subtilis in secretion of human lysozyme. Biochem Biophys Res Commun 145, 712–718. 2200 40 Miller GL & Golder RH (1950) Buffers of pH 2 to 12 for use in electrophoresis. Arch Biochem 29, 420–423. 41 Otwinowski Z & Minor W (1997) Processing of x-ray diffraction data collected in oscillation mode. In Macromolecular Crystallography, Part A (Carter CWJ & Sweet RM, eds), pp. 307–326. Academic Press, San Diego. 42 Vagin A & Teplyakov A (2000) An approach to multicopy search in molecular replacement. Acta Crystallogr D Biol Crystallogr 56, 1622–1624. 43 Collaborative Computational Project, Number 4 (1994) The CCP4 suite – programs for protein crystallography. Acta Crystallogr D Biol Crystallogr 50, 760–763. 44 Artymiuk PJ & Blake CCF (1981) Refinement of human lysozyme at 1.5 A resolution analysis of nonbonded and hydrogen-bond interactions. J Mol Biol 152, 737–762. 45 Murshudov GN, Vagin AA & Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53, 240–255. 46 Emsley P & Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60, 2126–2132. 47 Griko YV, Freire E, Privalov G, van Dael H & Privalov PL (1995) The unfolding thermodynamics of c-type lysozymes: a calorimetric study of the heat denaturation of equine lysozyme. J Mol Biol 252, 447–459. 48 Meiler J (2003) Proshift: protein chemical shift prediction using artificial neural networks. J Biomol NMR 26, 25–37. 49 Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD & Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In The Proteomics Protocols Handbook (Walker JM, ed.), pp. 571–607. Humana Press, Totowa, NJ. 50 Koradi R, Billeter M & Wuthrich K (1996) Molmol: a program for display and analysis of macromolecular structures. J Mol Graph 14, 51–55, 29–32. FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS