Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic Archaeon -: Evidence for Ca2+-induced folding
Crystal structure of unautoprocessed precursor of subtilisin from a hyperthermophilic Archaeon -: Evidence for Ca2+-induced folding
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DOI：
10.1074/jbc.m610137200
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发表时间：
2007-03-16
影响因子：
4.8
通讯作者：
Kanaya, Shigenori
中科院分区：
文献类型：
--
作者：
Tanaka, Shun-ichi;Saito, Kenji;Kanaya, Shigenori
在2.3埃分辨率下测定了来自超嗜热古菌Thermococcus kodakaraensis的前Tk-枯草杆菌蛋白酶活性位点突变体（pro-S324 A）的晶体结构。该蛋白的整体结构与细菌枯草杆菌蛋白酶-前肽复合物的结构相似，不同之处在于连接前肽和成熟结构域的肽键与活性位点接触，并且成熟结构域含有6个Ca 2+结合位点。Ca-1位点在细菌枯草杆菌蛋白酶中是保守的，但在自动加工之前形成，不像细菌枯草杆菌蛋白酶的相应位点。所有其他Ca 2+结合位点在pro-S324 A结构中是独特的，并且位于表面环。其中四个显然有助于中央α β α亚结构的成熟域的稳定性。CD光谱、1-苯胺基-8-萘磺酸荧光光谱和对胰凝乳蛋白酶消化的敏感性表明，pro-S324 A的构象在Ca 2+结合后从不稳定的熔融球状结构变为稳定的天然结构。另一个活性位点突变体pro-S324 C在Ca ~（2+）存在下被自动加工形成前肽-成熟结构域复合物。该蛋白的CD光谱表明pro-S324 C的结构在与Ca 2+结合时发生变化，类似于pro-S324 A，但在随后的自动加工后没有严重变化。这些结果表明，Tk-枯草杆菌蛋白酶的成熟过程是不同的细菌枯草杆菌蛋白酶的成熟结构域的折叠和完成折叠过程之前的自动加工的要求的Ca 2+。
The crystal structure of an active site mutant of pro-Tk-subtilisin (pro-S324A) from the hyperthermophilic archaeon Thermococcus kodakaraensis was determined at 2.3 angstrom resolution. The overall structure of this protein is similar to those of bacterial subtilisin-propeptide complexes, except that the peptide bond linking the propeptide and mature domain contacts with the active site, and the mature domain contains six Ca2+ binding sites. The Ca-1 site is conserved in bacterial subtilisins but is formed prior to autoprocessing, unlike the corresponding sites of bacterial subtilisins. All other Ca2+-binding sites are unique in the pro-S324A structure and are located at the surface loops. Four of them apparently contribute to the stability of the central alpha beta alpha substructure of the mature domain. The CD spectra, 1-anilino-8-naphthalenesulfonic acid fluorescence spectra, and sensitivities to chymotryptic digestion of this protein indicate that the conformation of pro-S324A is changed from an unstable molten globule-like structure to a stable native one upon Ca2+ binding. Another active site mutant, pro-S324C, was shown to be autoprocessed to form a propeptide-mature domain complex in the presence of Ca2+. The CD spectra of this protein indicate that the structure of pro-S324C is changed upon Ca2+ -binding like pro-S324A but is not seriously changed upon subsequent autoprocessing. These results suggest that the maturation process of Tk-subtilisin is different from that of bacterial subtilisins in terms of the requirement of Ca2+ for folding of the mature domain and completion of the folding process prior to autoprocessing.