Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon.
Tk-subtilisin from the hyperthermophiolic archaeon Thermococcus kodakaraensis matures from Pro-Tk-subtilisin upon autoprocessing and degradation of Tk-propeptide [Tanaka, S., Saito, K., Chon, H., Matsumura, H., Koga, Y., Takano, K., and Kanaya, S. (2007) J. Biol. Chem. 282, 8246-8255]. It requires Ca(2+) for folding and assumes a molten globule-like structure in the absence of Ca(2+) even in the presence of Tk-propeptide. Tk-subtilisin contains seven Ca(2+)-binding sites. Four of them (Ca2-Ca5) are located within a long loop, which mostly consists of a unique insertion sequence of this protein. To analyze the role of this Ca(2+)-binding loop, three mutant proteins, Deltaloop-Tk-subtilisin, DeltaCa2-Pro-S324A, and DeltaCa3-Pro-S324A, were constructed. These proteins were designed to remove the Ca(2+)-binding loop, Ca2 site, or Ca3 site of Pro-Tk-subtilisin or its active site mutant Pro-S324A. Far-UV CD spectra of these proteins refolded in the absence and presence of Ca(2+) indicated that Deltaloop-Tk-subtilisin completely lost the ability to fold into a native structure. In contrast, two other proteins retained this ability, although their refolding rates were greatly decreased compared to that of Pro-S324A. Determination of the crystal structures of these proteins purified in a Ca(2+)-bound form indicates that the structures of DeltaCa2-Pro-S324A and DeltaCa3-Pro-S324A are virtually identical to that of Pro-S324A, except that they lack the Ca2 and Ca3 sites, respectively, and the structure of the Ca(2+)-binding loop is destabilized. Nevertheless, these proteins were slightly more stable than Pro-S324A. These results suggest that the Ca(2+)-binding loop is required for folding of Tk-subtilisin but does not seriously contribute to the stabilization of Tk-subtilisin in a native structure.
Takeuchi
Yuki
Y
Department of Material and Life Science, Graduate School of Engineering, Osaka University,2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Tanaka
Shun-ichi
S
Matsumura
Hiroyoshi
H
Koga
Yuichi
Y
Takano
Kazufumi
K
Kanaya
Shigenori
S
D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D017931:DNA Primers; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D020860:Subtilisin / Q000235:genetics / Q000378:metabolism*; D019712:Thermococcus / Q000201:enzymology*; D014961:X-Ray Diffraction
D017931:DNA Primers; D020860:Subtilisin; D002118:Calcium