2ZWP

Crystal structure of Ca3 site mutant of Pro-S324A

Summary for 2ZWP

• Entry DOI: 10.2210/pdb2zwp/pdb
• Related: 2Z2Z 2ZWO
• Descriptor: Tk-subtilisin, CALCIUM ION (3 entities in total)
• Functional Keywords: subtilisin, thermococcus kodakaraensis, calcium ion, folding, calcium, hydrolase, protease, secreted, serine protease, zymogen
• Biological source: Thermococcus kodakaraensis
• Cellular location: Secreted: P58502
• Total number of polymer chains: 2
• Total formula weight: 83137.48

Authors

Takeuchi, Y.,Tanaka, S.,Matsumura, H.,Koga, Y.,Takano, K.,Kanaya, S. (deposition date: 2008-12-17, release date: 2009-06-23, Last modification date: 2024-10-09)

Primary citation

Takeuchi, Y.,Tanaka, S.,Matsumura, H.,Koga, Y.,Takano, K.,Kanaya, S.
Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon.
Biochemistry, 48:10637-10643, 2009

PubMed Abstract: 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.

PubMed: 19813760
DOI: 10.1021/bi901334b

Experimental method

X-RAY DIFFRACTION (2.4 Å)