Department of Material and Life Science, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
J Mol Biol. 2010 Jul 23;400(4):865-77. doi: 10.1016/j.jmb.2010.05.064. Epub 2010 Jun 1.
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.
Tk-SP 是一种来自 Thermococcus kodakaraensis 的超耐热枯草杆菌样丝氨酸蛋白酶，可自其前体（Pro-Tk-SP）经 N 端和 C 端肽段加工而来。Pro-Tk-SP 的活性位点突变体（缺失 C 端肽段的 ProN-Tk-S359A）的晶体结构在 2.0Å 分辨率下得以确定。ProN-Tk-S359A 由 N 端肽段、枯草杆菌蛋白酶和 β-发夹环结构域组成。两个 Ca²⁺离子与 β-发夹环结构域结合。无 β-发夹环结构域的 ProN-Tk-S359A 的整体结构与细菌前肽段-枯草杆菌蛋白酶复合物相似，只是它不含有 Ca²⁺离子。为了分析 Tk-SP 的 β-发夹环结构域的作用，我们构建了一系列 Tk-SP 的活性位点突变体（含或不含 β-发夹环结构域的 Tk-S359A/C 和 Tk-S359A/CDeltaJ）。Tk-S359C 和 Tk-S359CDeltaJ 在凝胶分析中均表现出蛋白酶活性，表明 β-发夹环结构域对于折叠或活性并非必需。然而，在 10mM CaCl₂存在下，用远紫外 CD 光谱法测定的 Tk-S359ADeltaJ 的 Tm 值比 Tk-S359A 低 29.4°C。用 10mM 乙二胺四乙酸处理后，Tk-S359A 的 Tm 值降低了 29.5°C，表明 β-发夹环结构域仅在结合 Ca²⁺的形式下对 Tk-S359A 的稳定起作用。Tk-SP 在大小和氨基酸序列上与 Pyrococcus furiosus、Thermococcus gammatolerans 和 Thermococcus onnurineus 的枯草杆菌样丝氨酸蛋白酶非常相似。我们提出，将 β-发夹环结构域连接到 C 末端是耐热蛋白适应高温环境的策略之一。
