Báo cáo khoa học: X-ray crystallography and structural stability of digestive lysozyme from cow stomach doc

X-ray crystallography and structural stability of digestive lysozyme from cow stomach Yasuhiro Nonaka 1 , Daisuke Akieda 1 , Tomoyasu Aizawa 1 , Nobuhisa Watanabe 1,2 , Masakatsu Kamiya 3 , Yasuhiro Kumaki 1 , Mineyuki Mizuguchi 4 , Takashi Kikukawa 1 , Makoto Demura 3 and Keiichi Kawano 1 1 Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo, Japan 2 Department of Biotechnology and Biomaterial Chemistry, Graduate School of Engineering, Nagoya University, Nagoya, Japan 3 Division of Molecular Life Science, Graduate School of Life Science, Hokkaido University, Sapporo, Japan 4 Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan 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 ver- tebrates, arthropods, and some other metazoa. It cata- lyzes 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 bacte- rial 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 com- ponent is then absorbed in the intestine. The acquisi- tion 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 (col- obus) 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 mate- rial, and digest the bacteria with lysozyme. According to phylogenetic analyses, each phyloge- netic group has independently adapted its defensive lysozyme for digestion [7,8]. Interestingly, common 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) doi:10.1111/j.1742-4658.2009.06948.x In ruminants, some leaf-eating animals, and some insects, defensive lyso- zymes have been adapted to become digestive enzymes, in order to digest bacteria in the stomach. Digestive lysozyme has been reported to be resis- tant 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 previ- ously reported for BSL2. The crystal structure of BSL2 suggested that negatively charged surfaces, a shortened loop and salt bridges could pro- vide structural stability, and thus resistance to pepsin. It is likely that BSL2 loses lytic activity at neutral pH because of adaptations to resist pepsin. 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 properties, e.g. low optimal pH and resistance to pro- tease, are shared by digestive lysozymes from different organisms [6,8–10]. Furthermore, ruminant and colo- bus 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 adap- tation 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 mech- anism underlying the acidic pH optimum [11]. The pK a values of the catalytic residues are lowered by neigh- boring residues, resulting in the acidic pH optimum. No experimental three-dimensional structure of ver- tebrate 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 stom- ach lysozyme 2 (BSL2), the most highly expressed lyso- zyme 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 P2 1 2 1 2 1 . The structure was refined at 1.5 A ˚ to an R-factor of 17.8% and an R-free of 22.1%. The average B-value for all protein atoms is 10.17 A ˚ 2 , and that for all main chain atoms is 9.25 A ˚ 2 . The electron density map was sufficiently clear to build a molecular model, and most of the side chain confor- mations 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 previ- ously 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 super- imposition of the main chain conformations of BSL2, human lysozyme, HEWL, and house fly midgut AB Fig. 1. (A) Ribbon model of BSL2 (Protein Data Bank ID: 2Z2F) in which a-helices are sequentially labeled from A to D. The struc- ture 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 C a conformation of BSL2 (red), human lyso- zyme (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]. Table 1. Data collection, processing and refinement statistics. Data collection Space group P2 1 2 1 2 1 Cell constants (A ˚ ) a 31.257 b 56.065 c 64.050 Resolution (A ˚ ) 50.00–1.50 (1.55–1.50) a No. observations 126 692 I ⁄ r(I) 28.085 (17.272) No. unique reflections 17833 (1662) R merge 0.046 (0.088) Completeness (%) 95.0 (90.7) Multiplicity 7.1 (7.1) Refinement data Resolution (A ˚ ) 17.94–1.50 No. reflections 16 849 R-factor 0.178 R free 0.221 Rmsd from ideal values Bond lengths (A ˚ ) 0.009 Bond angles (°) 1.261 a Values in parentheses are for the last resolution shell. Y. Nonaka et al. Structure and stability of bovine stomach lysozyme FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 2193 lysozyme. The rmsd between BSL2 and human lyso- zyme, calculated using the backbone atoms in the a-helices, is 0.38 A ˚ , that between BSL2 and HEWL is 0.35 A ˚ , and that between BSL2 and house fly lysozyme is 0.79 A ˚ . The backbone structure of BSL2 is closer to that of the vertebrate nondigestive lysozyme than to that of insect digestive lysozyme. 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 main- tain 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]. 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 (C m ), Gibbs free energies without denaturant (DG w ), 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 transi- tion temperatures (T m ) and unfolding enthalpy values (DH u ) at pH 2.0, obtained by thermal unfolding experi- ments using differential scanning calorimetry (DSC), are also summarized in Table 2. BSL2 unfolded at a higher temperature and had a greater DH u value, also indicating greater structural stability. Hydrogen exchange properties were monitored by 1D 1 H-NMR at pH 1.9, to compare the conforma- tional flexibilities of BSL2 and HEWL (Fig. 5). Gener- ally, 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 tryp- tophan residues whose peaks diminished rather slowly could exist in rigid and unexposed regions. Fig. 2. Bacteriolytic activities of BSL2 (gray bars) and HEWL (white bars) at different pH values, ionic strength 0.1, and 25 °C. The rela- tive activities are expressed by taking the activity of HEWL at pH 7.0 as 1.0. A B C Fig. 3. SDS ⁄ PAGE of pepsin-treated BSL2 and HEWL with (A) 0m M 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. Structure and stability of bovine stomach lysozyme Y. Nonaka et al. 2194 FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 num- ber 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 bac- terial 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 crys- tallographic analysis and catalytic activity experiments indicated that the catalytic residues have lower pK a values than those of HEWL, and thus the optimal pH is shifted to the acidic range [11]. Using the crystallo- graphic structures, we calculated the pK a values of the 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, f app , were plotted against the concentration of guanidinium hydrochlo- ride. The lines are the transition curves estimated by the nonlinear least squares method. Table 2. Thermodynamic parameters for guanidinium hydrochlo- ride-induced and thermal unfolding. pH 2.0 pH 6.0 BSL2 HEWL BSL2 HEWL Guanidinium hydrochloride-induced unfolding C m (M) 3.07 2.17 4.17 4.16 DG w (kJÆmol )1 ) 32.9 17.3 53.4 41.9 m (kJÆmol )1 M) 10.7 7.97 12.8 10.1 Thermal unfolding T m (K) 333.8 326.6 DH (kJÆmol )1 ) a 406.4 386.4 a The unfolding enthalpies at transition temperature T m . W64 A B W111 Normalized intensityNormalized intensity 11.0 10.8 10.6 10.4 10.2 p.p.m. 10.0 9.8 11.0 10.8 10.6 10.4 10.2 p.p.m. 10.0 9.8 W63 W34 W108 W108 W62 W63 W111 W123 Fig. 5. 1D 1 H-NMR spectra of (A) BSL2 and (B) HEWL in 95% H 2 O ⁄ 5% D 2 O (thick lines) and after 30, 60 or 120 min of hydro- gen–deuterium exchange in 100% D 2 O (thin lines). The spectra were acquired at pH 1.9. Y. Nonaka et al. Structure and stability of bovine stomach lysozyme FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 2195 catalytic residues Glu35 and Asp52 (numbering for HEWL), for BSL2 and other lysozymes, with prop- ka 2.0 [16]. The predicted pK a 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 pK a values for house fly lyso- zyme are well predicted. The calculated pK a 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 pK a , whereas the polarity of Thr110 reduces the pK a for house fly lysozyme. In the case of Asp52, the pK a is modulated by the hydrogen bond network. There are hydrogen bonds formed by Asp52, Asn46 and Asp48 in HEWL. House fly lysozyme has an aspara- gine at position 48, and the absence of the negative charge should reduce the pK a 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 pK a . However, Asp52 in BSL2 is more exposed to solvent than that in HEWL, and this raises the pK a . As a result, the calculated pK a 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 flexibil- ity of the polypeptide substrate [12,17,18]. The correla- tion between structural rigidity and stability has been reported for many proteins [19–22]. The high confor- mational 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 pro- tected, 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 HEWL. These results support the notion that confor- mational rigidity protects BSL2 from pepsin digestion. Because the house fly lysozyme is resistant to cathep- sin 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 con- formational rigidity often leads to a reduction in enzy- matic 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 posi- tively 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, how- ever, 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 struc- ture [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 Table 3. Comparison of structural parameters among lysozymes. BSL2 HEWL Human House fly No. of residues 129 129 130 122 No. of charged residues Negative 15 9 11 9 Positive 18 18 20 12 No. of salt bridges 4 3 5 3 No. of hydrogen bonds 125 122 125 109 Hydrogen bonds ⁄ residue 0.97 0.95 0.96 0.89 Structure and stability of bovine stomach lysozyme Y. Nonaka et al. 2196 FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 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 recom- binant BSL2 with a perfect sequence using the methylo- trophic 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 selec- tion and phenotypic screening were performed on a mini- mal 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 previ- ously 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 metha- nol 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 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 absor- bance 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 NH 4 HCO 3 and freeze-dried. Assay of lytic activity The lytic activities of BSL2 and HEWL against M. lys- odeikticus 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]. Lyso- zyme 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 absor- bance 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 (sit- ting drop) method, using 0.1 m sodium Hepes buffer at pH 7.5, containing 0.2 m NaCl and 30% 2-methyl-2,4-penta- nediol. The space group of the crystal was P2 1 2 1 2 1 , with cell dimensions a = 31.257 A ˚ , b = 56.065 A ˚ , and c = 64.050 A ˚ . There is one monomeric molecule in an asymmet- ric 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 Y. Nonaka et al. Structure and stability of bovine stomach lysozyme FEBS Journal 276 (2009) 2192–2200 ª 2009 The Authors Journal compilation ª 2009 FEBS 2197 acceptance of the detector. The limit at the edge of the detec- tor using an 80 mm crystal-to-film distance is approximately 1.5 A ˚ resolution. The structure was solved by the molecular replacement method, using the program molrep [42] pack- aged in ccp4 [43]. The structure of recombinant human lyso- zyme (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 func- tions 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 dis- tance. 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 A ˚ . The hydrogen bonds were detected using the what if web interface with the following criteria: maximal distances of 3.5 A ˚ for donor–acceptor and 2.5 A ˚ 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 spectro- polarimeter (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 concentra- tion 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 = DG w – mC, where DG and DG w 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, Northamp- ton, MA, USA), at a scan rate of 1.0 KÆmin )1 . Sample solution was prepared with reference buffer 50 mm glycine- HCl 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 scan. The DSC curves were analyzed to obtain the transi- tion temperatures (T m ) and unfolding enthalpies (DH u ) [47]. Hydrogen–deuterium exchange experiment Hydrogen–deuterium exchange was measured by 1D 1 H- NMR 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 D 2 O 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 1 H-NMR in the same buffer with 95% H 2 O ⁄ 5% D 2 O. The peaks of unexchangeable hydrogens were used to normalize inten- sity. 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 spectrophoto- metrically 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 Pro- motion 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 Biochemis- try and Biology (Jolles P, ed.), pp. 9–31. EXS, Basel. 2 Langer P (1974) Stomach evolution in the artiodactyla. 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