|Home | About | Journals | Submit | Contact Us | Français|
Crystallization of and preliminary crystallographic studies on an active-site mutant of pro-Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis were performed. The crystal was grown at 277 K by the sitting-drop vapour-diffusion method. Native X-ray diffraction data were collected to 2.3 Å resolution using synchrotron radiation from station BL41XU at SPring-8. The crystal belongs to the orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 Å. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient V M was calculated to be 2.6 Å3 Da−1 and the solvent content was 53.1%.
Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis is a member of the subtilisin family (Kannan et al., 2001 ; Pulido et al., 2006 ). Bacterial subtilisins such as subtilisin E, subtilisin BPN′ and subtilisin Carlsberg represent this family, which is one of six families of subtilisin-like serine proteases (subtilases; Siezen & Leunissen, 1997 ). Like bacterial subtilisins, Tk-subtilisin consists of a signal peptide (Met−93–Ala−70), propeptide (Gly−69–Leu−1) and mature domain (Gly1–Gly329). The mature domain of Tk-subtilisin contains three insertion sequences compared with those of bacterial subtilisins. This domain without insertion sequences is similar to those of bacterial subtilisins both in size and amino-acid sequence (amino-acid sequence identity of 45–46%).
Bacterial subtilisins are secreted into an external medium in a pro-form (pro-subtilisin) and are activated upon autoprocessing and degradation of the propeptide (Shinde & Inouye, 1996 ). The latter process is required to produce active subtilisin, because the propeptide remains tightly bound to the mature domain after autoprocessing and thereby inhibits its activity (Li et al., 1995 ; Huang et al., 1997 ; Yabuta et al., 2001 ). It has been proposed that the propeptides of bacterial subtilisins function not only as inhibitors of their cognate mature domains but also as intramolecular chaperones that facilitate folding of the mature domains (Eder et al., 1993 ; Shinde et al., 1997 ; Subbian et al., 2005 ). The mature domains alone are not folded into an active form but are folded into an inactive form with a molten globular-like structure in the absence of propeptides (Eder et al., 1993 ; Shinde & Inouye, 1995 ). Requirement of a propeptide for the maturation of its cognate mature domain has also been reported not only for other members of the subtilase family (Baier et al., 1996 ; Marie-Claire et al., 2001 ; Basak & Lazure, 2003 ), but also for other proteases (Silen & Agard, 1989 ; Smith & Gottesman, 1989 ; Winther & Sorensen, 1991 ; O’Donohue & Beaumont, 1996 ; Marie-Claire et al., 1999 ; Nirasawa et al., 1999 ).
Subtilases contain several Ca2+-binding sites, which vary in number (two to four) and location (Bode et al., 1987 ; Betzel et al., 1988 ; Gros et al., 1988 ; Smith et al., 1999 ). Subtilisin BPN′ is greatly destabilized upon removal of the high-affinity Ca2+-binding site (Voordouw et al., 1976 ). Pro-subtilisin E is folded and autoprocessed in the absence of Ca2+ (Yabuta et al., 2002 ). A subtilisin BPN′ derivative without a Ca2+-binding site exhibits Ca2+-independent activity (Gallagher et al., 1993 ; Strausberg et al., 1995 ). These results suggest that Ca2+ is not required for activity but is required for the stability of subtilisin.
Like bacterial subtilisins, Tk-subtilisin is maturated (activated) from pro-Tk-subtilisin upon autoprocessing and degradation of propeptide (Pulido et al., 2006 ). In this process, the propeptide is first autoprocessed to produce an inactive complex between the propeptide and the mature domain. Then, the propeptide, which is simultaneously both a potent inhibitor and 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, probably to produce its active conformation (Pulido et al., 2006 ). In the absence of Ca2+, pro-Tk-subtilisin is not maturated at all even at high temperatures. In addition, Tk-subtilisin does not require propeptide for folding of its mature domain, because the mature domain alone is refolded and exhibits Ca2+-dependent activity in the absence of propeptide (Pulido et al., 2006 ). These results suggest that the maturation process of Tk-subtilisin is different from those of bacterial subtilisins.
To understand the unique maturation process of pro-Tk-subtilisin, it is necessary to determine its crystal structure. However, pro-Tk-subtilisin is not fully stable in the presence of Ca2+ even at 277 K, especially when its concentration is high. Under these condition, pro-Tk-subtilisin is gradually converted to an active mature form, which is finally self-degraded. In this report, we constructed the active-site mutant of pro-Tk-subtilisin (pro-S255A), overproduced and purified the recombinant protein, crystallized it in complex with Ca2+ and performed preliminary X-ray crystallographic studies.
The gene encoding the active-site mutant of pro-Tk-subtilisin (pro-S255A) was amplified by PCR using the overlap extension method (Horton et al., 1990 ). The mutagenic primers were designed such that the codon for Ser255 (AGC) is changed to GCC for Ala. The derivative of pET25b (Novagen) for overproduction of pro-S255A was constructed as previously described for pro-Tk-subtilisin (Kannan et al., 2001 ). 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; Perkin–Elmer) and KOD DNA polymerase (Toyobo). The DNA sequence of the gene encoding pro-S255A was confirmed using an ABI Prism 310 DNA sequencer (Perkin–Elmer).
Overproduction of pro-S255A in Escherichia coli BL21-CodonPlus(DE3) was carried out as described previously for pro-Tk-subtilisin (Kannan et al., 2001 ). The cells were collected by centrifugation, suspended in 20 mM Tris–HCl pH 9.0, disrupted by sonication on ice and centrifuged at 30 000g for 30 min at 277 K. The pellet was dissolved in 20 mM Tris–HCl pH 9.0 containing 8 M urea and 5 mM EDTA and applied onto a HiTrap Q HP column (Pharmacia/GE Healthcare) equilibrated with the same buffer. The protein was eluted from the column with a linear gradient of 0–0.3 M NaCl. The fractions containing denatured pro-S255A, which eluted from the column at approximately 0.1 M NaCl, were collected and dialyzed against 20 mM Tris–HCl pH 7.0 containing 10 mM CaCl2 and 1 mM DTT for 5 d for refolding. The refolded pro-S255A protein was incubated at 353 K for 30 min and centrifuged at 30 000g for 30 min at 277 K. The resultant supernatant was finally loaded onto a Sephacryl S-200HR column (Pharmacia/GE Healthcare) equilibrated with 20 mM Tris–HCl pH 7.0 containing 10 mM CaCl2 and 0.05 M NaCl. The fractions containing pro-S255A were collected, dialyzed against 10 mM Tris–HCl pH 7.0 and concentrated using a Centricon ultrafiltration system (Millipore). The protein concentration was determined from UV absorption using a cell with an optical path length of 1 cm and an A 280 value of 1.25 for a 0.1% solution. The purity of the protein was confirmed by SDS–PAGE (Laemmli, 1970 ) followed by staining with Coomassie Brilliant Blue.
The crystallization conditions were initially screened using crystallization kits from Hampton Research (Crystal Screens I, II and Cryo) and Emerald Biostructures (Wizard I and II) with a TASCAL-1 semiautomatic protein crystallization system (Kentoku Industry Co. Ltd, Suita, Japan; Adachi et al., 2004 ). The conditions were surveyed using the sitting-drop vapour-diffusion method at 277 and 293 K. Drops were prepared by mixing 1 µl each of the protein solutions (approximately 10 mg ml−1 pro-S255A in 10 mM Tris–HCl pH 7.0) and the reservoir solution and were vapour-equilibrated against 100 µl reservoir solution. Crystals appeared after a few weeks using Crystal Screen I solution No. 33 (4.0 M sodium formate). The crystallization conditions were not further optimized as cube-shaped single crystals suitable for X-ray diffraction analysis were obtained from the initial screening.
A crystal of pro-S255A was mounted on a CryoLoop (Hampton Research), adding Paratone-N (Hampton Research) as cryoprotectant, and then flash-frozen in a nitrogen-gas stream at 100 K. X-ray diffraction data were collected at a wavelength of 1.0 Å on beamline BL41XU at SPring-8, Japan. A total of 180 images were recorded with an exposure time of 15 s per image and an oscillation angle of 1.0°. Diffraction images were indexed, integrated and scaled using the HKL-2000 program suite (Otwinowski & Minor, 1997 ).
Comparison of the amino-acid sequence of pro-Tk-subtilisin with those of various subtilases indicates that Asp46, His84 and Ser255 form the catalytic triad of Tk-subtilisin. Therefore, the active-site mutant of pro-Tk-subtilisin (pro-S255A) was designed such that Ser255 is replaced by Ala. Upon induction for overproduction, pro-S255A accumulated in the cells in inclusion bodies like pro-Tk-subtilisin. It was solubilized in 8 M urea, purified in the presence of 8 M urea and refolded by removing urea in the presence of Ca2+. The protein was refolded in the presence of Ca2+ because it has been suggested that the mature domain of pro-Tk-subtilisin requires Ca2+ to attain its active conformation (Pulido et al., 2006 ). The refolded protein was further purified by gel-filtration column chromatography to give a single band on SDS–PAGE (Fig. 1 ). The amount of the protein purified from 1 l culture was typically 10 mg.
The molecular weight of pro-S255A estimated from gel-filtration column chromatography (45 kDa) was nearly identical to that estimated from SDS–PAGE, indicating that pro-S255A exists in a monomeric form. Unlike pro-Tk-subtilisin, which is rapidly maturated in the presence of Ca2+ at 353 K, pro-S255A was not autoprocessed at all but remained intact under these conditions (data not shown), indicating that Tk-subtilisin is completely inactivated by the mutation. Pro-Tk-subtilisin was refolded in the presence of Ca2+, dialyzed against Ca2+-free buffer and crystallized. However, the number of the Ca2+ ions bound to the protein used for crystallization was determined to be six by atomic absorption spectrometry (Jarrel-Ash A-8500 Mark II), indicating that at least six Ca2+ ions bind to pro-S255A too tightly to be removed by dialysis.
Crystals appeared after a few weeks and grew to maximum dimensions of 0.43 × 0.35 × 0.30 mm after one month (Fig. 2 ). The crystals diffracted to 2.3 Å. A total of 124 455 measured reflections were merged into 18 765 unique reflections with an R merge of 4.8%. The crystals belong to the primitive orthorhombic space group I222, with unit-cell parameters a = 92.69, b = 121.78, c = 77.53 Å. Table 1 summarizes the data-collection statistics. Based on the molecular weight and the space group, it was assumed that the crystal contains one protein molecule per asymmetric unit, giving a V M value of 2.6 Å3 Da−1 and a solvent content of 53.1%. These values are within the ranges frequently observed for protein crystals (Matthews, 1968 ), suggesting that this crystal is suitable for structural determination. It has also been found that six calcium ions bind to pro-S255A at the loop regions. We are currently solving this structure.
The synchrotron-radiation experiments were performed at the BL41XU at SPring-8 with the approval of the Japan Synchrotron Radiation Research Institute (JASRI; Proposal No. 2005B1766). This work was supported in part by a Grant-in-Aid for the 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 (NEDO) of Japan.