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J. Biol. Chem., Vol. 282, Issue 11, 8246-8255, March 16, 2007

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Crystal Structure of Unautoprocessed Precursor of Subtilisin from a Hyperthermophilic Archaeon

EVIDENCE FOR Ca²⁺-INDUCED FOLDING^*

Shun-ichi Tanaka, Kenji Saito, Hyongi Chon, Hiroyoshi Matsumura^¶, Yuichi Koga, Kazufumi Takano^¶, and Shigenori Kanaya¹

From the Departments of Material and Life Science and Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan, and ^¶CREST (Sosho Project), JST, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan

Received for publication, October 30, 2006 , and in revised form, December 21, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
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 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
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 terminus 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 BPN', 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 (7–9). 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 (11–13) 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 Ca²⁺ 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 Ca²⁺ from site 1 (24–26). Prosubtilisin E is folded and autoprocessed in the absence of Ca²⁺ (27). Deletion of the loop forming site 1 of subtilisin BPN', followed by directed evolution and selection for increased stability, results in a Ca²⁺-independent subtilisin mutant with native-like activity (25, 28, 29). These results suggest that Ca²⁺ is not required for activity or folding of subtilisin but is required for stability. However, the number and location of the Ca²⁺ binding sites vary greatly for other members of the subtilase family. For example, thermitase has three Ca²⁺ 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 Ca²⁺-dependent activity. These results suggest that the role of the Ca²⁺ ions varies for different subtilases.

Tk-subtilisin² from the hyperthermophilic archaeon Thermococcus kodakaraensis consists of signal peptide (Met^-24–Ala^-1), propeptide (Gly¹–Leu⁶⁹), and mature domain (Gly⁷⁰–Gly³⁹⁸) (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 Ca²⁺ for activity and is therefore not matured at all even at high temperatures in the absence of Ca²⁺. 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 Ca²⁺-bound form. However, pro-Tk-subtilisin is not fully stable in the presence of Ca²⁺ 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 Ca²⁺-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 Ca²⁺ 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
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 Ser³²⁴ (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 x 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 0.1 M for pro-S324A and pro-S324C and 0.2 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 Ca²⁺-free form, these proteins (25 µM) were dialyzed against 20 mM Tris-HCl (pH 7.0) at 4 °C. For refolding of pro-S324A and pro-S324C in a Ca²⁺-bound form, these proteins (25 µM) were dialyzed against 20 mM Tris-HCl (pH 7.0) containing 10 mM CaCl₂ and 1 mM DTT at 4 °C for 5 days, incubated at 80 °C for 30 min, and centrifuged at 30,000 x 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 CaCl₂ and 50 mM NaCl. The fractions containing the protein were collected and dialyzed against 20 mM Tris-HCl (pH 7.0) containing 10 mM CaCl₂.

Pro-Tk-subtilisin, mat-Tk-subtilisin, and Tk-propeptide were overproduced and purified as described previously (2). mat-Tk-subtilisin^*, which represents a Ca²⁺-bound active form of mat-Tk-subtilisin, was prepared by incubating pro-Tk-subtilisin at 80 °C for 90 min in the presence of Ca²⁺ 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 A₂₈₀ 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^-1 cm^-1 for tyrosine and 5,225 M^-1 cm^-1 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 µl) contains 50 mM CAPS-NaOH (pH 9.5), 5 mM CaCl₂, 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^-1 cm^-1 by automatic UV spectrophotometer (Beckman model DU640). One unit of enzymatic activity was defined as the amount of the enzyme that produced 1 µmol 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 CaCl₂ for protein in a Ca²⁺-bound form and 20 mM Tris-HCl (pH 7.0) for protein in a Ca²⁺-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 cm² dmol^-1, 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 µM) and ANS (50 µM) were dissolved in 20 mM Tris-HCl (pH 7.0) either in the presence or absence of 10 mM CaCl₂. 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 µM) 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 CaCl₂ and either in the presence or absence of 0.3 µM 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 µl 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 Ca²⁺-bound Form—Pro-S324A purified in a Ca²⁺-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 = 92.69, b = 121.78, c = 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 subtilisin-propeptide complex (Protein Data Bank entry 1SCJ [PDB] ) 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 Ca²⁺ ions coordinated by six or seven ligands were observed, refining with full occupancy in the electron density (2F_o - 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

View larger version (63K): [in this window] [in a new window]   FIGURE 1. Three-dimensional structure of pro-S324A. Stereo view of the main-chain folding of pro-S324A in a Ca2+-bound form (A) and superposition of this structure on the structure of S221C-subtilisin E-propeptide complex (B) are shown. For the pro-S324A structure, the propeptide domain (Asn4–Lue69) and mature domain (Gly70–Gly398) are colored red and green, respectively. The loops formed by three major insertion sequences (Gly70–Pro82, Pro207–Asp226, and Gly346–Ser358) are colored blue, magenta, and orange, respectively. Two active site residues (Asp115 and His153) and Ala324, 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 Ca2+ 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 Ca2+ ions, is colored gray. Three active site residues (Asp32, His64, and Cys221) are indicated by stick models. The labels for this structure are shown in parentheses.

The mature domain consists of a seven-stranded parallel -sheet (1m–3m and 5m–8m), 4m-strand, an anti-parallel -sheet (9m and 10m), and 10 -helices (1m–10m). Of them, the seven-stranded parallel -sheet and two -helices (5m and 8m) form a core structure. As in all members of the subtilase family, three active site residues, Asp¹¹⁵, His¹⁵³, and Ser³²⁴, form a catalytic triad in Tk-subtilisin. In the present structure, Ser³²⁴ is replaced by Ala. Two hydrogen bonds are formed between His¹⁵³ N¹ and Asp¹¹⁵ O¹ and between His¹⁵³ N¹ and Asp¹¹⁵ O². These two hydrogen bonds are also observed in the subtilisin E-propeptide complex structure (22). The pro-S324A structure has one disulfide bond between Cys¹³² and Cys¹⁴⁷, 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 Gly⁷⁰–Pro⁸², Pro²⁰⁷–Asp²²⁶, and Gly³⁴⁶–Ser³⁵⁸. 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 Ca²⁺ binding sites (Fig. 1A). The overall structure of pro-S324A in a Ca²⁺-bound 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 Å for 65 C atoms from Thr⁵ to Leu⁶⁹ in the propeptide domain and 0.6 Å for 264 C atoms from Ala⁸³ to Gln³⁹⁴, except Ser¹⁰⁷-Val¹⁰⁸, Ala¹²⁷-Asn¹²⁸, Gly¹³⁸–Arg¹⁴⁵, Pro²⁰⁷–Asp²²⁶, Gln³⁴³–Lys³⁴⁷, Thr³⁵³–Asn³⁶⁰, and Thr³⁷⁶–Trp³⁷⁸, in the mature domain. However, this structure was considerably different from that of the subtilisin E-propeptide complex in the positions of Ca²⁺ binding sites as described below.

View larger version (129K): [in this window] [in a new window]   FIGURE 2. A stereo view of electron density around the active site (A) and Ca-1 site (B) of pro-S324A. In A, the 2Fo - Fc map contoured at the 1.5 level is shown. In B, the 2Fo - Fc maps contoured at the 2.0 and 5.0 levels are shown in blue and magenta, respectively. The active site residues, the nitrogen and oxygen atoms, and the Ca2+ ions are indicated as in Fig. 1.

The other five Ca²⁺ 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 (Gly²⁰⁶–Glu²²⁹) (Fig. 1). This Ca²⁺ binding loop is rich in Asp and is mostly formed by a unique insertion sequence of Tk-subtilisin. The Ca²⁺ 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 Ca²⁺ 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 (Asp²¹⁴, Asp²¹⁶, and Asp²²²) for coordination.

The Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ binding loop. As a result, they do not contain any Ca²⁺ 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 Ca²⁺ ions instead of the propeptide. This may be the reason why the mature domain of Tk-subtilisin requires Ca²⁺ instead of the propeptide for folding.

The Ca-6 site is located at a surface loop between the 9m- and 10m-helices. In this site, the Ca²⁺ 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 Ca²⁺ ion cannot bind to this loop. Insertion of the three amino acid residues (Thr³⁷⁶–Trp³⁷⁸) widens this loop, and the introductions of two aspartic acid residues (Asp³⁷² and Asp³⁷⁹) into this loop, which provide ligands for Ca²⁺ binding, may be responsible for the formation of the Ca-6 site in pro-Tk-subtilisin. Binding of the Ca²⁺ ion to this site may be responsible for tight packing of the C-terminal 9m- and 10m-helices. Because the 10m-helix interacts with the N-terminal loop region between the 1m- and 2m-helices, this site may be important for both thermal and proteolytic stability of Tk-subtilisin.

Ca²⁺-induced Folding—The crystallographic studies indicate that the structure of pro-S324A in a Ca²⁺-bound form represents a native structure of this protein. To examine whether the conformation of pro-Tk-subtilisin is changed upon Ca²⁺ binding, the far-UV and near-UV CD spectra of pro-S324A in a Ca²⁺-free and Ca²⁺-bound form were measured. The far-UV and near-UV CD spectra of pro-S324A in a Ca²⁺-free form were nearly identical to those of pro-Tk-subtilisin in a Ca²⁺-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 Ca²⁺-bound form was considerably different from that of pro-S324A in a Ca²⁺-free form (Fig. 4A). The far-UV CD spectrum of pro-S324A in a Ca²⁺-free form gave a broad trough with a single minimum around at 208 nm, whereas that of pro-S324A in a Ca²⁺-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 Ca²⁺-bound form was considerably different from that of pro-S324A in a Ca²⁺-free form (Fig. 4B). These results suggest that the conformation of pro-S324A is greatly changed upon Ca²⁺ 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 Ca²⁺-free form was changed to that in a Ca²⁺-bound form in the presence of 10 mM CaCl₂, and the far-UV CD spectrum of pro-S324A in a Ca²⁺-bound form was changed to that in a Ca²⁺-free form upon treatment with 50 mM EDTA.

ANS Fluorescence Spectroscopy—To obtain more information on the Ca²⁺-induced conformational change of pro-Tk-subtilisin, 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 Ca²⁺-bound form was greatly reduced as compared with that of pro-S324A in a Ca²⁺-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 Ca²⁺ but is folded into a native structure in the presence of Ca²⁺.

Protein Stability—To examine whether Ca²⁺ 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 Ca²⁺-free form was almost fully degraded even at an enzyme/substrate ratio of 1:1000, whereas pro-S324A in a Ca²⁺-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 Ca²⁺ binding. It has been reported that Ca²⁺ 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 Ca²⁺, instead of the propeptide, induces folding of the mature domain into a highly stable functional structure.

View larger version (30K): [in this window] [in a new window]   FIGURE 3. Ca2+ binding sites. The structures of the Ca-1 (A), Ca-2 to Ca-5 (B), and Ca-6 (D) sites are shown. The Ca2+ ions and water molecules are represented by spheres. The residues that bind to the Ca2+ ions are labeled. 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, Asp115, is indicated by a stick model.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. CD spectra. Shown are far-UV (left) and near-UV (right) CD spectra of pro-S324A in a Ca2+-free form (thin line), pro-S324A in a Ca2+-bound form (thick line), and the mat-S324C*-propeptide complex (broken line). Spectra were measured at 20 °C as described under "Experimental Procedures."

View larger version (18K): [in this window] [in a new window]   FIGURE 5. ANS fluorescence spectra. The ANS fluorescence spectra of pro-S324A in a Ca2+-free form (thin line), pro-S324A in a Ca2+-bound form (thick line), and mat-S324C*-propeptide complex (broken line) are shown. These spectra were measured at 20 °C as described under "Experimental Procedures."

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 Ca²⁺, 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 Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ nor that refolded in the absence of Ca²⁺ 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.

View larger version (110K): [in this window] [in a new window]   FIGURE 6. Susceptibility of pro-S324A to chymotryptic digestion. Pro-S324A in a Ca2+-free form (lanes 1–4) and pro-S324A in a Ca2+-bound form (lanes 5 and 6) were digested with chymotrypsin at 30 °C for 1 h in 20 mM Tris-HCl (pH 7.0) at enzyme/substrate ratios (by weight) of 1:10,000 (lane 2), 1:1000 (lane 3), 1:100 (lane 4), and 1:10 (lane 6) and subjected to 15% SDS-PAGE. The protein was stained with CBB. M, low molecular weight marker kit (Amersham Biosciences); lane 1, undigested pro-S324A in a Ca2+-free form; lane 5, undigested pro-S324A in a Ca2+-bound form. The molecular mass of each standard protein is indicated beside the gel.

View larger version (95K): [in this window] [in a new window]   FIGURE 7. Comparison of the refolding yield of mat-Tk-subtilisin and mat-S324A in the presence and absence of Tk-propeptide. For refolding of mat-Tk-subtilisin, the unfolded protein (30 µM) was diluted 100-fold with buffer A (50 mM Tris-HCl (pH 7.0) containing 1 mM DTT) (lane 1), Ca2+ buffer (50 mM Tris-HCl (pH 7.0) containing 1 mM DTT and 10 mM CaCl2)(lane 2), or Ca2+/propeptide buffer (50 mM Tris-HCl (pH 7.0) containing 1 mM DTT, 10 mM CaCl2, and Tk-propeptide (0.3 µM)) (lane 3) at 4 °C, incubated at 80 °C for 30 min, and subjected to 15% SDS-PAGE. For refolding of mat-S324A, the unfolded protein (30 µM) was diluted 100-fold with buffer A (lane 7), Ca2+ buffer (lane 8), propeptide buffer (50 mM Tris-HCl (pH 7.0) containing 1 mM DTT and Tk-propeptide (0.3 µM)) (lane 9), or Ca2+/propeptide buffer (lane 10) at 4 °C, digested with chymotrypsin at 30 °C for 1 h at an enzyme/substrate ratio of 1:100 (w/w), and subjected to 15% SDS-PAGE. Lane 4, undigested mat-Tk-subtilisin*; lane 5, mat-Tk-subtilisin* digested with chymotrypsin as mentioned above; lane 6, undigested mat-S324A. The protein was stained with CBB. M, low molecular weight marker kit. The molecular mass of each standard protein is indicated beside the gel. The upper and lower arrows indicate the positions of mat-Tk-subtilisin* or mat-S324A and Tk-propeptide, respectively.

View larger version (57K): [in this window] [in a new window]   FIGURE 8. SDS-PAGE of pro-S324A and pro-S324C refolded in the presence of Ca2+. Pro-S324A (lane 1) and pro-S324C (lane 2) refolded in the presence of Ca2+ were analyzed by 15% SDS-PAGE. The arrows indicate the positions of pro-S324A, mat-S324C*, and Tk-propeptide from the top to the bottom. The molecular mass of each standard protein is indicated beside the gel.

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 Ca²⁺ 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

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION CONCLUSION REFERENCES

 
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 Ca²⁺ 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 Ca²⁺ binding and is almost fully completed prior to autoprocessing. The mature domain of pro-S324A contains six Ca²⁺ 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 Ca²⁺ binding. These results suggest that the role of Ca²⁺ and propeptide for maturation greatly varies for different subtilases.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2E1P) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported in part by a Grant-in-aid for National Project on Protein Structural and Functional Analyses and by an Industrial Technology Research Grant Program from the New Energy and Industrial Technology Development Organization of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel./Fax: 81-6-6879-7938; E-mail: kanaya{at}mls.eng.osaka-u.ac.jp.

² The abbreviations used are: Tk-subtilisin, subtilisin from T. kodakaraensis; pro-Tk-subtilisin, Tk-subtilisin in a pro-form (Gly¹–Gly³⁹⁸); Tk-propeptide, propeptide of Tk-subtilisin (Gly¹–Leu⁶⁹); mat-Tk-subtilisin, Tk-subtilisin in a mature form (Gly⁷⁰–Gly³⁹⁸); mat-Tk-subtilisin^*, a Ca²⁺-bound active form of mat-Tk-subtilisin; pro-S324A (S324C), pro-Tk-subtilisin with the Ser³²⁴ Ala (Ser³²⁴ Cys) mutation; mat-S324A(S324C), mat-Tk-subtilisin with the Ser³²⁴ Ala (Ser³²⁴ Cys) mutation; mat-S324C^*, mat-Tk-subtilisin^* with the Ser³²⁴ Cys mutation; CBB, Coomassie Brilliant Blue; DTT, dithiothreitol; ANS, 1-anilino-8-naphthalenesulfonic acid.

	ACKNOWLEDGMENTS

 
The synchrotron radiation experiments were performed at the BL41XU beamline of SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (Proposal 2005B1766). We thank Drs. T. Inoue and Y. Kai for support in X-ray crystallography and for helpful discussions. We thank M. Pulido for critical reading of the manuscript.

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