Tk-subtilisin from the hyperthermophilic archaeon Thermococcus kodakaraensis
is a member of the subtilisin family (Kannan et al.
; Pulido et al.
). 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
), propeptide (Gly−69
) and mature domain (Gly1
). 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.
; Huang et al.
; Yabuta et al.
). 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.
; Shinde et al.
; Subbian et al.
). 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.
; 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.
; Marie-Claire et al.
; 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.
; Nirasawa et al.
Subtilases contain several Ca2+
-binding sites, which vary in number (two to four) and location (Bode et al.
; Betzel et al.
; Gros et al.
; Smith et al.
). Subtilisin BPN′ is greatly destabilized upon removal of the high-affinity Ca2+
-binding site (Voordouw et al.
). Pro-subtilisin E is folded and autoprocessed in the absence of Ca2+
(Yabuta et al.
). A subtilisin BPN′ derivative without a Ca2+
-binding site exhibits Ca2+
-independent activity (Gallagher et al.
; Strausberg et al.
). 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.
). 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.
). 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.
). 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.