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首页> 外文期刊>Journal of Molecular Biology >Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability.
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Crystal structure of a subtilisin homologue, Tk-SP, from Thermococcus kodakaraensis: requirement of a C-terminal beta-jelly roll domain for hyperstability.

机译：枯草蛋白酶同源物Tk-SP的晶体结构，来自热球菌（Kodkaraensis）：超稳定性需要C端β-果冻卷结构域。

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摘要

Tk-SP is a hyperthermostable subtilisin-like serine protease from Thermococcus kodakaraensis and is autoprocessed from its precursor (Pro-Tk-SP) with N- and C-propeptides. The crystal structure of the active-site mutant of Pro-Tk-SP lacking C-propeptide, ProN-Tk-S359A, was determined at 2.0 A resolution. ProN-Tk-S359A consists of the N-propeptide, subtilisin, and beta-jelly roll domains. Two Ca(2+) ions bind to the beta-jelly roll domain. The overall structure of ProN-Tk-S359A without the beta-jelly roll domain is similar to that of the bacterial propeptide:subtilisin complex, except that it does not contain Ca(2+) ions. To analyze the role of the beta-jelly roll domain of Tk-SP, we constructed a series of the active-site mutants of Tk-SP with (Tk-S359A/C) and without (Tk-S359A/CDeltaJ) beta-jelly roll domain. Both Tk-S359C and Tk-S359CDeltaJ exhibited protease activities in gel assay, indicating that the beta-jelly roll domain is not required for folding or activity. However, the T(m) value of Tk-S359ADeltaJ determined by far-UV CD spectroscopy in the presence of 10-mM CaCl(2) was lower than that of Tk-S359A by 29.4 degrees C. The T(m) value of Tk-S359A was decreased by 29.5 degrees C by the treatment with 10 mM ethylenediaminetetraacetic acid, indicating that the beta-jelly roll domain contributes to the stabilization of Tk-S359A only in a Ca(2+)-bound form. Tk-SP highly resembles subtilisin-like serine proteases from Pyrococcus furiosus, Thermococcus gammatolerans, and Thermococcus onnurineus in size and amino acid sequence. We propose that attachment of a beta-jelly roll domain to the C-terminus is one of the strategies of the proteins from hyperthermophiles to adapt to high-temperature environment.
机译：Tk-SP是得自Kodakaraensis的Thermcoccus kodakaraensis的一种超耐高温的枯草杆菌蛋白酶样丝氨酸蛋白酶，可从其前体（Pro-Tk-SP）中分别用N和C肽进行自动加工。缺乏C肽ProN-Tk-S359A的Pro-Tk-SP活性位点突变体的晶体结构以2.0 A的分辨率确定。 ProN-Tk-S359A由N-前肽，枯草杆菌蛋白酶和β-果冻卷结构域组成。两个Ca（2+）离子绑定到β-果冻卷域。没有β-果冻卷结构域的ProN-Tk-S359A的总体结构类似于细菌前肽：枯草杆菌蛋白酶复合物，只是它不包含Ca（2+）离子。为了分析Tk-SP的β-果冻卷结构域的作用，我们构建了一系列带有（Tk-S359A / C）和没有（Tk-S359A / CDeltaJ）β-果冻的Tk-SP活性位点突变体滚动域。 Tk-S359C和Tk​​-S359CDeltaJ在凝胶测定中均显示蛋白酶活性，表明折叠或活性不需要β-果冻卷结构域。但是，在10mM CaCl（2）存在下，通过远紫外CD光谱法测定的Tk-S359ADeltaJ的T（m）值比Tk-S359A的T（m）值低29.4摄氏度。通过用10 mM乙二胺四乙酸处理，Tk-S359A降低了29.5摄氏度，表明β-果冻卷结构域仅以Ca（2+）结合形式有助于Tk-S359A的稳定化。 Tk-SP的大小和氨基酸序列与酷热球菌，嗜热球菌和嗜热球菌的枯草杆菌蛋白酶样丝氨酸蛋白酶高度相似。我们建议将β-果冻卷结构域连接到C端是来自超嗜热菌的蛋白质适应高温环境的策略之一。

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