Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon.
Requirement of a unique Ca(2+)-binding loop for folding of Tk-subtilisin from a hyperthermophilic archaeon.
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DOI：
10.1021/bi901334b
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发表时间：
2009-10
期刊：
影响因子：
2.9
通讯作者：
Yuki Takeuchi;Shun-ichi Tanaka;H. Matsumura;Y. Koga;K. Takano;S. Kanaya
中科院分区：
文献类型：
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作者：
Yuki Takeuchi;Shun-ichi Tanaka;H. Matsumura;Y. Koga;K. Takano;S. Kanaya
来自超嗜热古菌Thermococcus kodakaraensis的Tk-枯草杆菌蛋白酶在Tk-前肽的自动加工和降解后从Pro-Tk-枯草杆菌蛋白酶成熟[Tanaka，S.，齐藤，K.，Chon，H.，Matsumura，H.，古贺，Y.，高野，K.，和Kanaya，S.（2007）J.Biol.Chem.282，8246-8255]。它需要Ca（2+）来折叠，并且即使在存在Tk-前肽的情况下，在不存在Ca（2+）的情况下也呈现熔融的球状结构。TK-枯草杆菌蛋白酶含有7个Ca（2+）结合位点。其中四个（Ca 2-Ca 5）位于一个长环内，该长环主要由该蛋白的独特插入序列组成。为了分析该Ca（2+）结合环的作用，构建了三种突变蛋白，Deltaloop-Tk-subtilisin、DeltaCa 2-Pro-S324 A和DeltaCa 3-Pro-S324 A。设计这些蛋白质以去除Pro-Tk-枯草杆菌蛋白酶或其活性位点突变体Pro-S324 A的Ca（2+）结合环、Ca 2位点或Ca 3位点。在Ca（2+）存在和不存在的情况下，这些蛋白质重折叠的远紫外CD光谱表明，Deltaloop-Tk-枯草杆菌蛋白酶完全丧失了折叠成天然结构的能力。与此相反，其他两种蛋白质保留了这种能力，虽然它们的重折叠速率大大降低相比，Pro-S324 A。以Ca（2+）结合形式纯化的这些蛋白质的晶体结构的测定表明，DeltaCa 2-Pro-S324 A和DeltaCa 3-Pro-S324 A的结构与Pro-S324 A的结构几乎相同，除了它们分别缺少Ca 2和Ca 3位点，并且Ca（2+）结合环的结构不稳定。然而，这些蛋白质比Pro-S324 A稍微更稳定。这些结果表明，Ca（2+）结合环是Tk-枯草杆菌蛋白酶折叠所必需的，但对Tk-枯草杆菌蛋白酶在天然结构中的稳定性没有重大贡献。
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.