Crystal Structure of Unautoprocessed Precursor of Subtilisin from a Hyperthermophilic Archaeon

The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3Å resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central αβα substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca2+ binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca2+ for folding of the mature domain and completion of the folding process prior to autoprocessing.

Subtilisin is a serine protease with broad substrate specificity. It is a member of the subtilase family (1) and is widely distributed in various organisms, including bacteria and archaea. These subtilisins are synthesized in a precursor form, in which a signal peptide and a propeptide are attached to the N termi-nus of the mature domain (1,2). The mechanism by which subtilisins are folded and matured has been most extensively studied for bacterial subtilisins, such as subtilisin E, subtilisin BPNЈ, and subtilisin Carlsberg. According to these studies, subtilisins are first secreted into an external medium in a pro form (prosubtilisin) with the assistance of signal peptide, and activated upon autoprocessing and degradation of propeptide (3). Degradation of propeptide is necessary for the mature domain to function as an active enzyme, because the propeptide continues to bind tightly to the mature domain after autoprocessing and thereby inhibits its activity (4 -6). It has been proposed that propeptides of subtilisins function not only as inhibitors of their cognate mature domains but also as intramolecular chaperones that facilitate folding of the mature domains after secretion (7)(8)(9). The mature domains alone are not an active form but an inactive form with a molten globule-like structure in the absence of propeptides (7,10). Dependence on the propeptide for maturation of its cognate mature domain has been reported not only for other members of the subtilase family (11)(12)(13) but also for other proteases (14 -19).
The crystal structures of bacterial subtilisins are available not only for the active mature form (20,21) but also for the inactive complex between the propeptide and mature domain (22,23). Comparison of these structures indicates that the folding process of the mature domain is almost fully completed prior to degradation of the propeptide. These structures also indicate that bacterial subtilisins have two Ca 2ϩ binding sites, site 1 (site A) and site 2 (site B), both of which are located far from the active site. Sites 1 and 2 have high and low binding affinity, respectively. The enzyme is greatly destabilized with respect to both thermal denaturation and autodegradation upon removal of Ca 2ϩ from site 1 (24 -26). Prosubtilisin E is folded and autoprocessed in the absence of Ca 2ϩ (27). Deletion of the loop forming site 1 of subtilisin BPNЈ, followed by directed evolution and selection for increased stability, results in a Ca 2ϩ -independent subtilisin mutant with native-like activity (25,28,29). These results suggest that Ca 2ϩ is not required for activity or folding of subtilisin but is required for stability. However, the number and location of the Ca 2ϩ binding sites vary greatly for other members of the subtilase family. For example, thermitase has three Ca 2ϩ binding sites (30), proteinase K (31), and Ak.1 protease (32) have four sites, and sphericase has five sites (33), which are located far from the active site but do not necessarily correlate to site 1 or site 2 of bacterial subtilisins. In addition, various subtilases, such as sphericase (33), cell envelope protease (34), and psychrophilic subtilisin (35), have been reported to exhibit Ca 2ϩ -dependent activity. These results suggest that the role of the Ca 2ϩ ions varies for different subtilases.
Tk-subtilisin 2 from the hyperthermophilic archaeon Thermococcus kodakaraensis consists of signal peptide (Met Ϫ24 -Ala Ϫ1 ), propeptide (Gly 1 -Leu 69 ), and mature domain (Gly 70 -Gly 398 ) (2). The mature domain of Tk-subtilisin contains three major insertion sequences as compared with those of bacterial subtilisins. Other than that, the sequence is similar to those of bacterial subtilisins both in size and amino acid sequence (amino acid sequence identities of 45-46%). Like bacterial subtilisins, Tk-subtilisin is matured from pro-Tk-subtilisin upon autoprocessing and degradation of the propeptide. In this process, the propeptide is autoprocessed at first to produce an inactive complex between the propeptide and mature domain. Then the propeptide, which is a potent inhibitor and, at the same time, a good substrate of the mature domain, is degraded by the mature domain to produce active enzyme. However, unlike bacterial subtilisins, Tk-subtilisin requires Ca 2ϩ for activity and is therefore not matured at all even at high temperatures in the absence of Ca 2ϩ . In addition, the propeptide of pro-Tk-subtilisin is not required for folding of the mature domain, although it is involved in folding of the mature domain in an auxiliary manner. These results suggest that the maturation process of Tk-subtilisin is different from that proposed for bacterial subtilisins. To understand this unique maturation process, it is necessary to determine the crystal structure of pro-Tk-subtilisin in a Ca 2ϩ -bound form. However, pro-Tksubtilisin is not fully stable in the presence of Ca 2ϩ even at 4°C, especially when its concentration is high. In this condition, pro-Tk-subtilisin is gradually converted to an active mature form, which is finally self-degraded.
We have recently succeeded in crystallizing the active site mutant of pro-Tk-subtilisin, pro-S324A, in a Ca 2ϩ -bound form and performed preliminary x-ray diffraction studies (36). In this report, we determined the crystal structure of this protein, which represents the unautoprocessed precursor structure of subtilisin, at 2.3 Å resolution. We also showed that the conformation of pro-S324A is considerably changed from a molten globule-like structure to a native one upon Ca 2ϩ binding. Furthermore, we constructed another active site mutant pro-S324C and characterized it biochemically. Based on these results, we discuss a unique maturation process of Tk-subtilisin.

EXPERIMENTAL PROCEDURES
Construction of Plasmids-The gene encoding pro-S324C was amplified by PCR using overlap extension method (37). The mutagenic primers were designed such that the codon for Ser 324 (AGC) is changed to TGC for Cys. The pET25b derivative for overproduction of pro-S324A (36) was used as a template. The pET25b derivatives for overproduction of pro-S324C and mat-S324A were constructed as previously described for pro-Tk-subtilisin and mat-Tk-subtilisin, respectively (2). All DNA oligomers for PCR were synthesized by Hokkaido System Science (Sapporo, Japan). PCR was performed in 25 cycles using a thermal cycler (Gene Amp PCR System 2400; PerkinElmer Life Sciences) and KOD DNA polymerase (Toyobo). The DNA sequences of the genes encoding pro-S324C and mat-S324A were confirmed by ABI Prism 310 DNA sequencer (PerkinElmer Life Sciences).
Overproduction and Purification of the Mutant Proteins-Overproduction of pro-S324A, pro-S324C, and mat-S324A in Escherichia coli BL21-codonPlus(DE3) (Stratagene) was carried out as described previously for pro-Tk-subtilisin (38). The cells were collected by centrifugation, suspended in 20 mM Tris-HCl (pH 9.0), disrupted by sonication on ice, and centrifuged at 30,000 ϫ g for 30 min at 4°C. The pellet was dissolved in 20 mM Tris-HCl (pH 9.0) containing 8 M urea and 5 mM EDTA and applied to a HiTrap Q HP column (GE Healthcare) equilibrated with the same buffer. The protein was eluted from the column at NaCl concentration of ϳ0.1 M for pro-S324A and pro-S324C and ϳ0.2 M for mat-S324A by linearly increasing the NaCl concentration from 0 to 0.3 M. The fractions containing the protein were collected and refolded as described below. For refolding of pro-S324A, pro-S324C, and mat-S324A in a Ca 2ϩfree form, these proteins (25 M) were dialyzed against 20 mM Tris-HCl (pH 7.0) at 4°C. For refolding of pro-S324A and pro-S324C in a Ca 2ϩ -bound form, these proteins (25 M) were dialyzed against 20 mM Tris-HCl (pH 7.0) containing 10 mM CaCl 2 and 1 mM DTT at 4°C for 5 days, incubated at 80°C for 30 min, and centrifuged at 30,000 ϫ g for 30 min at 4°C. The resultant supernatant was loaded onto a Sephacryl S-200HR column (GE Healthcare) equilibrated with 20 mM Tris-HCl (pH 7.0) containing 10 mM CaCl 2 and 50 mM NaCl. The fractions containing the protein were collected and dialyzed against 20 mM Tris-HCl (pH 7.0) containing 10 mM CaCl 2 .
Pro-Tk-subtilisin, mat-Tk-subtilisin, and Tk-propeptide were overproduced and purified as described previously (2). mat-Tk-subtilisin*, which represents a Ca 2ϩ -bound active form of mat-Tk-subtilisin, was prepared by incubating pro-Tksubtilisin at 80°C for 90 min in the presence of Ca 2ϩ as described previously (2) and used without further purification.
The protein concentration was determined from UV absorption using a cell with an optical path length of 1 cm and A 280 value for 0.1% (1 mg/ml) solution of 1.25 for pro-Tk-subtilisin, pro-S324A, and pro-S324C; 1.47 for mat-Tk-subtilisin and mat-S324A; and 0.21 for Tk-propeptide. These values were calculated by using absorption coefficients of 1,526 M Ϫ1 cm Ϫ1 for tyrosine and 5,225 M Ϫ1 cm Ϫ1 for tryptophan at 280 nm (39). The purity of the protein was confirmed by SDS-PAGE (40), followed by staining with Coomassie Brilliant Blue (CBB). For N-terminal amino acid sequencing, the protein visualized with CBB staining was transferred to a nitrocellulose membrane (blotting). The-N-terminal amino acid sequence was determined using a Procise 491 automated protein sequencer (PerkinElmer Life Sciences).
Enzymatic Activity-The enzymatic activity was determined by using N-succinyl-AAPF-p-nitroanilide (Sigma) as a substrate at 80°C. The reaction mixture (100 l) contains 50 mM CAPS-NaOH (pH 9.5), 5 mM CaCl 2 , and 2 mM N-succinyl-AAPF-p-nitroanilide. The amount of p-nitroaniline released from the substrate was determined from the absorption at 410 nm with an absorption coefficient of 8,900 M Ϫ1 cm Ϫ1 by automatic UV spectrophotometer (Beckman model DU640). One unit of enzymatic activity was defined as the amount of the enzyme that produced 1 mol of p-nitroaniline/min. The specific activity was defined as the enzymatic activity/mg of protein.
Circular Dichroism Spectroscopy-The far-UV (200 -260 nm) and near-UV (250 -320 nm) CD spectra of the protein were measured by a J-725 automatic spectropolarimeter (Japan Spectroscopic Co.) at 20°C. The buffer was 20 mM Tris-HCl (pH 7.0) containing 10 mM CaCl 2 for protein in a Ca 2ϩ -bound form and 20 mM Tris-HCl (pH 7.0) for protein in a Ca 2ϩ -free form. The protein concentration and optical path length were 0.15 mg/ml and 2 mm for far-UV CD spectra and 1.0 mg/ml and 1 cm for near-UV CD spectra, respectively. The mean residue ellipticity, [], which has the unit of degrees cm 2 dmol Ϫ1 , was calculated by using an average amino acid molecular weight of 110.
1-Anilino-8-naphthalenesulfonic Acid (ANS) Fluorescence Spectroscopy-Binding of ANS (Sigma) to the protein was analyzed by measuring the fluorescence of ANS at 20°C. The protein (1 M) and ANS (50 M) were dissolved in 20 mM Tris-HCl (pH 7.0) either in the presence or absence of 10 mM CaCl 2 . The protein was incubated at 20°C for 4 h prior to the measurement of ANS fluorescence. The excitation wavelength was 380 nm, and the emission was monitored from 400 to 600 nm. The spectrum obtained in the absence of the protein was used as blank.
Analysis of Folding Efficiency-mat-Tk-subtilisin or mat-S324A (30 M) was first unfolded by incubating it for 1 h at 25°C in 20 mM Tris-HCl (pH 7.0) containing 6 M guanidine HCl. The protein was refolded by 100-fold dilution with 50 mM Tris-HCl (pH 7.0) containing 1 mM DTT, either in the presence or absence of 10 mM CaCl 2 and either in the presence or absence of 0.3 M Tk-propeptide at 4°C. Then refolded mat-Tk-subtilisin was incubated at 80°C for 30 min, and refolded mat-S324A was digested with chymotrypsin at 30°C for 1 h at an enzyme/substrate ratio of 1:100 (w/w) after incubation at 4°C for 1 day. Finally, 112 l of trichloroacetic acid was added to 1 ml of the reaction mixture to precipitate the proteins. The resultant precipitates were washed with 70% acetone and analyzed by 15% SDS-PAGE.
Crystallization of Pro-S324A in Ca 2ϩ -bound Form-Pro-S324A purified in a Ca 2ϩ -bound form was dialyzed against 10 mM Tris-HCl (pH 7.0), concentrated to 10 mg/ml using an ultrafiltration system Centricon (Millipore Corp.), and crystallized using 4 M sodium formate as described previously (36). X-ray diffraction studies on the resultant crystals indicated that these belonged to the space group I222 (unit cell parameters a ϭ 92.69, b ϭ 121.78, c ϭ 77.53 Å) and contained single protein molecules per asymmetric unit.

X-ray Diffraction Data Collection and Structure
Determination-x-ray diffraction data were collected at a wavelength of 1.0 Å with the beam line BL41XU station at SPring-8, Japan. Diffraction data were indexed, integrated, and scaled using the HKL2000 program suite (41). The crystal structure was solved by the molecular replacement method using MOLREP (42) in the CCP4 program suite. The 2.1 Å structure of subtilisinpropeptide complex (Protein Data Bank entry 1SCJ) was used as a starting model. Model building and refinement of the structure were completed using the programs O (43) and CNS (44). After molecular replacement, six Ca 2ϩ ions coordinated by six or seven ligands were observed, refining with full occupancy in the electron density (2F o Ϫ F c ). Progress in the structure refinement was evaluated at each stage by the free R factor and by inspection of stereochemical parameters calculated by the program PROCHECK (45). The Ramachandran plot produced by PROCHECK showed that almost all residues in the structure were in the favored region. The statistics for data collection and refinement are presented in Table 1. The figures were prepared by PyMol (available on the World Wide Web at www.pymol.org).

RESULTS AND DISCUSSION
Overall Three-dimensional Structure of Pro-S324A-The crystal structure of pro-S324A in a Ca 2ϩ -bound form was determined at 2.3 Å resolution. The asymmetric unit of the crystal structure consists of one protein molecule containing 395 of 398 residues, with three residues at the N terminus miss- ing, 270 water molecules, and six calcium ions. This structure is composed of the propeptide domain (Asn 4 -Leu 69 ) and mature domain (Gly 70 -Gly 398 ). The overall structure of pro-S324A in a Ca 2ϩ -bound form is shown in Fig. 1A. The propeptide domain folds into a compact structure with a four-stranded antiparallel ␤-sheet (␤1p-␤4p) and two ␣-helices (␣1p and ␣2p). This ␤-sheet packs tightly to the two nearly parallel ␣-helices (␣6m and ␣7m) located at the surface of the mature domain. Glu 61 and Asp 63 of the propeptide domain also bind to the N termini of these two ␣-helices to form helix caps. In addition, the linker peptide between the propeptide and mature domain runs through the active site cleft in a substrate/ product-like manner, with the Leu 69 -Gly 70 peptide bond (cleavage site) docked close to Ala 324 , which is substituted for the catalytic serine residue ( Fig. 2A). Leu 69 and Gly 70 occupy subsites S1 and S1Ј of the mature domain, respectively. Leu 69 oxygen forms a hydrogen bond with Ala 324 nitrogen. The ␤5pstrand of the propeptide domain interacts with ␤4m-strand of the mature domain to form an antiparallel ␤-sheet in the vicin-ity of the active site. A similar structure has been reported for the linker peptide of prokumamolisin, which is a member of a different subtilase family (46).
The mature domain consists of a seven-stranded parallel ␤-sheet (␤1m-␤3m and ␤5m-␤8m), ␤4mstrand, an anti-parallel ␤-sheet (␤9m and ␤10m), and 10 ␣-helices (␣1m-␣10m). Of them, the sevenstranded parallel ␤-sheet and two ␣-helices (␣5m and ␣8m) form a core structure. As in all members of the subtilase family, three active site residues, Asp 115 , His 153 , and Ser 324 , form a catalytic triad in Tk-subtilisin. In the present structure, Ser 324 is replaced by Ala. Two hydrogen bonds are formed between His 153 N ␦1 and Asp 115 O ␦1 and between His 153 N ␦1 and Asp 115 O ␦2 . These two hydrogen bonds are also observed in the subtilisin E-propeptide complex structure (22). The pro-S324A structure has one disulfide bond between Cys 132 and Cys 147 , which are the only cysteine residues in the pro-S324A sequence. A disulfide bond is formed at the same position in subtilisin S39 and subtilisin S41 from psychrophilic Bacillus (35) and sphericase from mesophilic Bacillus (33) but is not formed at any position in bacterial subtilisins. It remains to be determined whether this disulfide bond contributes to the high stability of Tk-subtilisin. Comparison of the amino acid sequence of pro-Tk-subtilisin with those of bacterial subtilisins based on the pro-S324A structure indicates that the pro-Tk-subtilisin sequence contains three major insertion sequences. They are Gly 70 -Pro 82 , Pro 207 -Asp 226 , and Gly 346 -Ser 358 . These insertion sequences form a loop at the protein surface, and two of them (the first and second one) are responsible for the formation of Ca 2ϩ binding sites (Fig. 1A). The overall structure of pro-S324A in a Ca 2ϩbound form is compared with that of subtilisin E-propeptide complex (22) in Fig. 1B. The former and latter structures represent those before and after cleavage of the propeptide, respectively. Comparison of these structures indicates that the main chain fold of pro-S324A is similar to that of the subtilisin E-propeptide complex, except that the N terminus of the mature domain moves away from the active site in the subtilisin E-propeptide complex structure, and the structure of the mature domain of pro-S324A contains three additional loops formed by the insertion sequences. These two structures could be superimposed with root mean square deviation values of 1.8 and mature domain (Gly 70 -Gly 398 ) are colored red and green, respectively. The loops formed by three major insertion sequences (Gly 70 -Pro 82 , Pro 207 -Asp 226 , and Gly 346 -Ser 358 ) are colored blue, magenta, and orange, respectively. Two active site residues (Asp 115 and His 153 ) and Ala 324 , which is substituted for the active site serine residue, are indicated by yellow stick models, in which the oxygen and nitrogen atoms are colored red and blue, respectively. Six Ca 2ϩ ions are shown in cyan spheres. N and C, N and C terminus, respectively. For the subtilisin E-propeptide complex, the entire structure, including two Ca 2ϩ ions, is colored gray. Three active site residues (Asp 32 , His 64 , and Cys 221 ) are indicated by stick models. The labels for this structure are shown in parentheses.
Å for 65 C␣ atoms from Thr 5 to Leu 69 in the propeptide domain and 0.6 Å for 264 C␣ atoms from Ala 83 to Gln 394 , except Ser 107 -Val 108 , Ala 127 -Asn 128 , Gly 138 -Arg 145 , Pro 207 -Asp 226 , Gln 343 -Lys 347 , Thr 353 -Asn 360 , and Thr 376 -Trp 378 , in the mature domain. However, this structure was considerably different from that of the subtilisin E-propeptide complex in the positions of Ca 2ϩ binding sites as described below.
Ca 2ϩ Binding Sites-Six Ca 2ϩ binding sites were identified in the mature domain of pro-S324A. This number, which was the highest among those reported for other subtilases to date, was consistent with that determined by atomic absorption spec-trometry (36). Only one of them (the Ca-1 site) was conserved in the structures of bacterial subtilisins, whereas others were unique in the pro-S324A structure. Four of these unique Ca 2ϩ binding sites were located at the surface loop, which was formed by the second insertion sequence of Tk-subtilisin. The structural details for these Ca 2ϩ binding sites are presented in Table 2.
The 2F o Ϫ F c map of pro-S324A around the Ca-1 site is shown in Fig.  2B as a representative. This site correlates to the high affinity site (site 1 or site A) of bacterial subtilisins. In this site, the Ca 2ϩ ion is heptacoordinated with six amino acid residues (Fig. 3A). All of these residues, except Ile 168 , which is replaced by Thr in one bacterial subtilisin sequence, are fully conserved in various bacterial subtilisin sequences. However, it has been proposed for bacterial subtilisins that one of these residues, Gln 2 , cannot make a direct coordination with the Ca 2ϩ ion in an unautoprocessed precursor form, because of the large distance between the high affinity site and active site (22,47). This means that the high affinity site of bacterial subtilisins is formed only when the peptide bond between the propeptide and mature domain is cleaved and the N-terminal region of the mature domain moves away from the active site. Hence, the unautoprocessed form of bacterial subtilisins is much less stable than the autoprocessed complex (6,27). In contrast, the corresponding residue of Tk-subtilisin, Gln 84 , can coordinate with Ca-1 in an unautoprocessed precursor form. This is clearly due to the presence of the loop formed by the insertion sequence (Gly 70 -Pro 82 ), which also exists as an N-terminal extension of the mature domain after autoprocessing. These results suggest that, unlike bacterial subtilisins, the conformation of pro-Tk-subtilisin is not seriously changed upon autoprocessing.
The other five Ca 2ϩ binding sites are unique to Tk-subtilisin and do not correlate to any site so far identified in various subtilases. Of them, four sites (Ca-2 to Ca-5) are located at a long external loop between ␣6m-helix and ␤5m-strand (Gly 206 -Glu 229 ) (Fig. 1). This Ca 2ϩ binding loop is rich in Asp and is mostly formed by a unique insertion sequence of Tk-subtilisin. The Ca 2ϩ ions are hexacoordinated with four residues and two water molecules in the Ca-2 site, six residues in the Ca-3 site, and four residues and one water molecule in the Ca-4 site and heptacoordinated with four residues and two water molecules in the Ca-5 site (Fig. 3B). The Ca 2ϩ ions bound to the Ca-3 and Ca-4 sites are located very close to each other, at a distance of 4.2 Å, and share the three aspartic acid residues (Asp 214 , Asp 216 , and Asp 222 ) for coordination.
The Ca 2ϩ binding loop apparently contributes to the stabilization of an ␣␤␣ substructure, which consists of ␣6m-helix, ␤5m-strand, and ␣7m-helix, because this loop interacts with the ␣7m-helix (Fig. 3C). In addition, this Ca 2ϩ binding loop probably stabilizes the central seven-stranded parallel ␤-sheet and thereby the core structure, because the Ca-5 site is located between the ␤5m and ␤1m-strands, both of which are constituents of the central seven-stranded parallel ␤-sheet. Bacterial subtilisins also have ␣␤␣ substructures (Fig. 1B). However, these structures greatly differ from that of Tk-subtilisin, because they lack a Ca 2ϩ binding loop. As a result, they do not contain any Ca 2ϩ binding site. It has been proposed that the stabilization of the ␣␤␣ substructure by the propeptide domain is crucial for subtilisin folding (47). However, in the case of Tk-subtilisin, the ␣␤␣ substructure is stabilized by the Ca 2ϩ ions instead of the propeptide. This may be the reason why the mature domain of Tk-subtilisin requires Ca 2ϩ instead of the propeptide for folding.
The Ca-6 site is located at a surface loop between the ␣9mand ␣10m-helices. In this site, the Ca 2ϩ ion is hexacoordinated with five residues and one water molecule (Fig. 3D). Bacterial subtilisins contain a similar surface loop at the same position. However, the Ca 2ϩ ion cannot bind to this loop. Insertion of the three amino acid residues (Thr 376 -Trp 378 ) widens this loop, and the introductions of two aspartic acid residues (Asp 372 and Asp 379 ) into this loop, which provide ligands for Ca 2ϩ binding, may be responsible for the formation of the Ca-6 site in pro-Tksubtilisin. Binding of the Ca 2ϩ ion to this site may be responsible for tight packing of the C-terminal ␣9mand ␣10m-helices. Because the ␣10m-helix interacts with the N-terminal loop region between the ␣1mand ␣2m-helices, this site may be important for both thermal and proteolytic stability of Tk-subtilisin.
Ca 2ϩ -induced Folding-The crystallographic studies indicate that the structure of pro-S324A in a Ca 2ϩ -bound form represents a native structure of this protein. To examine whether the conformation of pro-Tk-subtilisin is changed upon Ca 2ϩ binding, the far-UV and near-UV CD spectra of pro-S324A in a Ca 2ϩ -free and Ca 2ϩ -bound form were measured. The far-UV and near-UV CD spectra of pro-S324A in a Ca 2ϩ -free form were nearly identical to those of pro-Tk-subtilisin in a Ca 2ϩ -free form (data not shown), suggesting that the conformation of pro-Tk-subtilisin is not seriously changed by the mutation. However, the far-UV CD spectrum of pro-S324A in a Ca 2ϩ -bound form was considerably different from that of pro-S324A in a Ca 2ϩ -free form (Fig. 4A). The far-UV CD spectrum of pro-S324A in a Ca 2ϩ -free form gave a broad trough with a single minimum around at 208 nm, whereas that of pro-S324A in a Ca 2ϩ -bound form gave a trough with a double minimum around at 210 and 222 nm. Likewise, the near-UV CD spectrum of pro-S324A in a Ca 2ϩ -bound form was considerably different from that of pro-S324A in a Ca 2ϩ -free form (Fig.  4B). These results suggest that the conformation of pro-S324A is greatly changed upon Ca 2ϩ binding, such that the helical content of the protein increases and the environment of the aromatic residues is altered. Pro-S324A changes its conformation in a reversible manner, because the far-UV CD spectrum of pro-S324A in a Ca 2ϩ -free form was changed to that in a Ca 2ϩbound form in the presence of 10 mM CaCl 2 , and the far-UV CD spectrum of pro-S324A in a Ca 2ϩ -bound form was changed to that in a Ca 2ϩ -free form upon treatment with 50 mM EDTA.
ANS Fluorescence Spectroscopy-To obtain more information on the Ca 2ϩ -induced conformational change of pro-Tksubtilisin, pro-S324A was analyzed for its binding ability to ANS. ANS is known to bind more effectively to the protein in a partially folded state than to that in a fully folded or unfolded state (48). As shown in Fig. 5, ANS fluorescence of pro-S324A in a Ca 2ϩ -bound form was greatly reduced as compared with that of pro-S324A in a Ca 2ϩ -free form. These results, as well as the CD spectra of pro-S324A, strongly suggest that this protein is folded into a molten globule-like structure in the absence of Ca 2ϩ but is folded into a native structure in the presence of Ca 2ϩ .
Protein Stability-To examine whether Ca 2ϩ is required not only for folding but also for stabilization of pro-Tk-subtilisin, the susceptibility of pro-S324A to chymotryptic digestion was analyzed. When this protein was incubated with chymotrypsin at 30°C for 1 h in 20 mM Tris-HCl (pH 7.0) at an enzyme/substrate ratio of 1:10 -1:10,000 (w/w), pro-S324A in a Ca 2ϩ -free form was almost fully degraded even at an enzyme/substrate ratio of 1:1000, whereas pro-S324A in a Ca 2ϩ -bound form was not degraded at all even at an enzyme/substrate ratio of 1:10 (Fig. 6). These results suggest that pro-S324A is greatly stabilized upon Ca 2ϩ binding. It has been reported that Ca 2ϩ is required for stability of bacterial subtilisins (24). However, this stabilization effect has only been analyzed for the mature form. The conformation of the unautoprocessed precursor of bacterial subtilisins has been reported to be significantly different from that of the autoprocessed complex, because the unautoprocessed precursor of bacterial subtilisins exists in a molten globule-like state with a high content of solvent-accessible hydrophobic surface area and is a proteolytically unstable conformer (10). Thus, the maturation process of pro-Tk-subtilisin is different from that of bacterial prosubtilisins, because Ca 2ϩ , instead of the propeptide, induces folding of the mature domain into a highly stable functional structure. Role of Propeptide-When the mature domain of Tk-subtilisin (mat-Tk-subtilisin) was refolded by dilution with the buffer containing Ca 2ϩ in the presence or absence of Tk-propeptide, followed by the incubation at 80°C for 30 min, the refolding yield was ϳ1 and 100% in the absence and presence of an equal molar concentration of Tk-propeptide, respectively (Fig. 7,  lanes 2 and 3). At the lower Tk-propeptide concentrations, this yield increased as the concentration of Tk-propeptide increased (data not shown). The specific activity of the refolded protein was comparable with that of mat-Tk-subtilisin* regardless of the refolding yield. These results indicate that mat-Tksubtilisin is converted to mat-Tksubtilisin* with a native structure In C, a stereo view of the central ␣␤␣ substructure consisting of the ␣6m-helix, ␤5m-strand, and ␣7m-helix and the ␤1m-strand are shown in a ribbon drawing. One of the active site residues, Asp 115 , is indicated by a stick model.
upon Ca 2ϩ binding even in the absence of Tk-propeptide, but this yield greatly increases when Tk-propeptide is added in trans. However, these results do not necessarily indicate that Tk-propeptide exhibits a chaperon function, because a possibility that the propeptide prevents degradation of preactivated mat-Tk-subtilisin by inhibiting the mat-Tk-subtilisin* activity cannot be ruled out. To examine this possibility, the active site mutant of mat-Tk-subtilisin, mat-S324A, was constructed. If the propeptide does not exhibit any chaperon function, mat-S324A will be almost fully refolded into an inactive native structure upon Ca 2ϩ binding even in the absence of Tk-propeptide.
Upon overproduction, mat-S324A accumulated in an insoluble form in the E. coli cells, solubilized by 8 M urea, purified to give a single band on SDS-PAGE, and refolded by removing urea, like the wild-type protein. The production level of mat-S324A was nearly identical to that of the wild-type protein, and 5 mg of the purified protein was obtained from 1 liter of culture. The Ca 2ϩ -induced folding of mat-S324A was analyzed as described for mat-Tk-subtilisin, except that the refolded pro-tein with a nonnative structure was removed by degradation with chymotrypsin instead of mat-Tk-subtilisin*. The chymotryptic digestion was carried out in a condition in which pro-S324A in a Ca 2ϩ -bound form (Fig. 6) and mat-Tk-subtilisin* (Fig. 7, lane 5) are not significantly degraded, whereas pro-S324A in a Ca 2ϩ -free form is fully degraded (Fig. 6). In this condition, mat-S324A refolded in the absence of Ca 2ϩ was fully degraded by chymotrypsin, regardless of whether it was refolded in the presence of Tk-propeptide (Fig. 7, lanes 7 and 9). This result indicates that mat-S324A is not refolded into a native structure in the absence of Ca 2ϩ . In contrast, mat-S324A was refolded into a chymotrypsin-resistant form, which represents a Ca 2ϩ -bound form with a native structure, with a yield of 20% in the presence of Ca 2ϩ , even in the absence of Tk-propeptide (Fig. 7, lane 4). This yield increased to nearly 100% when an equal molar concentration of Tk-propeptide was added in trans (Fig. 7, lane 10). These results suggest that the propeptide of pro-Tk-subtilisin is required to increase the refolding yield of the mature domain not only by preventing degradation of a preactivated form of the protein but also by assisting the folding of the mature domain as an intramolecular chaperon.
Conformation of Autoprocessed Protein-It has been reported for a bacterial subtilisin that the mutation of the active site serine residue to Cys greatly reduces the enzymatic activity, such that the protein is autoprocessed but the propeptide is not further degraded by the mature domain (49). It has also been reported that the conformation of prosubtilisin is significantly changed upon autoprocessing (10). To examine whether this mutation blocks the maturation process of pro-Tk-subtilisin prior to the degradation of the propeptide and the conformation of pro-Tk-subtilisin is changed upon autoprocessing, the mutant protein pro-S324C was constructed.
Upon overproduction, pro-S324C accumulated in an insoluble form in the E. coli cells, solubilized by 8 M urea, purified, and refolded by removing urea in the absence or presence of Ca 2ϩ , like pro-S324A. The production level of pro-S324C was nearly identical to that of the wild-type protein, and 10 mg of the purified protein was obtained from 1 liter of culture. Pro-S324C refolded in the presence of Ca 2ϩ was eluted from the gel filtration column as a single peak at the position where pro-S324A was eluted. Nevertheless, it gave two bands on SDS-PAGE with the molecular masses of 35 and 10 kDa (Fig. 8, lane 2). These proteins were identified as mat-S324C and Tk-propeptide, respectively, by N-terminal amino acid sequence determination. In contrast, pro-S324C refolded in the absence of Ca 2ϩ gave a single band on SDS-PAGE at the position where pro-S324A is migrated (Fig. 8, lane 1). Neither pro-S324C refolded in the presence of Ca 2ϩ nor that refolded in the absence of Ca 2ϩ exhibited enzymatic activity. These results indicate that pro-S324C is autoprocessed into the propeptide and mature domain, but the propeptide continues to bind tightly to the mature domain to form a stable complex. The mature domain of pro-S324C (mat-S324C*) probably exhibits a very weak activity, which is sufficient to promote autoprocessing but is not sufficient for subsequent degradation of the propeptide, like the corresponding bacterial subtilisin mutant. The autoprocessed form of pro-S324C will be designated as mat-S324C*-propeptide complex hereafter.
The far-UV and near-UV CD spectra of the mat-S324C*propeptide complex were almost identical to those of pro-S324A in a Ca 2ϩ -bound form (Fig. 4). Likewise, ANS fluorescence of the mat-S324C*-propeptide complex was nearly identical to that of pro-S324A in a Ca 2ϩ -bound form (Fig. 5). The far-UV and near-UV CD spectra and ANS fluorescence of pro-S324C in a Ca 2ϩ -free form were indistinguishable from those of pro-S324A in a Ca 2ϩ -free form. Furthermore, pro-S324C in a Ca 2ϩ -free form was highly sensitive to chymotryptic digestion, whereas the mat-S324C*-propeptide complex was highly resistant to it, like pro-S324A. These results indicate that the conformation of pro-S324C is considerably changed upon Ca 2ϩ binding but is not seriously changed upon subsequent autoprocessing. Thus, the folding process of pro-Tk-subtilisin is almost fully completed prior to autoprocessing.
Adaptation to High Temperatures-Comparison of the crystal structures of pro-S324A and the subtilisin E-propeptide complex indicates that the amino acid residues forming an interface between the propeptide and mature domain are relatively well conserved, whereas those forming a hydrophobic core of the propeptide are not. The number of the hydrophobic residues located at the core of the propeptide of Tk-subtilisin (11 residues) is much higher than that of subtilisin E (six), indicating that the propeptide of Tk-subtilisin is highly more stable   than that of subtilisin E. In addition, as mentioned above, the mature domain of Tk-subtilisin is highly stabilized by Ca 2ϩ binding. The source organism of Tk-subtilisin, T. kodakaraensis, grows most optimally at 90°C (50). Tk-subtilisin probably adapts to a hyperthermic environment by increasing stability of both the propeptide and mature domain.

CONCLUSION
In this study, the crystal structure of pro-S324A, which represents an unautoprocessed precursor form of subtilisin, was determined at 2.3 Å resolution. In addition, the effects of Ca 2ϩ binding and autoprocessing on structure and stability of pro-Tk-subtilisin mutants were biochemically analyzed. These results show that the folding of unautoprocessed precursor of Tk-subtilisin is induced by Ca 2ϩ binding and is almost fully completed prior to autoprocessing. The mature domain of pro-S324A contains six Ca 2ϩ binding sites, all of which contribute to the stabilization of the protein. Bacterial subtilisins require a propeptide for folding, because their structure is unstable in the absence of propeptide. In contrast, Tk-subtilisin does not require propeptide for folding, probably because its structure is highly stabilized by Ca 2ϩ binding. These results suggest that the role of Ca 2ϩ and propeptide for maturation greatly varies for different subtilases.