Requirement of a Unique Ca2+-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 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 Ca2+ for folding and assumes it molten globule-like structure in the absence in the absence of Ca2+ even in the presence of Tk-propeptide Tk-subtilisin contains structure Ca2+-binding sites. Four of them (Ca2-Ca5) are located within a long loop, which Mostly Consists Of I unique insertion sequence of this protein. To analyze the role of this Ca2+-binding loop, three mutant proteins, Delta loop-Tk-subtilisin, Delta Ca2-Pro-S324A. and Delta Ca3-Pro-S324A, were constructed. These proteins were designed to remove the Ca2+-binding loop, Ca2 site, or Ca3 site orPro- Tk-subtilism or its active site mutant Pro-S324A Far-UVCD spectra of these proteins refolded in the absence and presence of Ca2+ indicated that Delta loop-Tk-subtilisin completely lost the ability to fold into it 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 Ca2+-bound form indicates that the structures of Delta Ca2-Pro-S324A and AC.O-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 Ca2+-binding loop is required for folding of Tk-subtilisin bill does not seriously contribute to the stabilizatioin of Tk-subtilisin in it native structure.