Formation of the high-affinity calcium binding site in Pro-subtilisin E with the insertion sequence IS1 of Pro-Tk-subtilisin.
Formation of the high-affinity calcium binding site in Pro-subtilisin E with the insertion sequence IS1 of Pro-Tk-subtilisin.
复制标题
使用 Pro-Tk-枯草杆菌蛋白酶的插入序列 IS1 在枯草杆菌蛋白酶原 E 中形成高亲和力钙结合位点。
作者：
Uehara;R.;Angkawidjaja;C.;Koga;Y.;and Kanaya;S.
枯草杆菌蛋白酶 E 是通过前肽的自动加工和降解从其无活性前体枯草杆菌蛋白酶原 E 中激活的。枯草杆菌蛋白酶 E 有两个钙结合位点，即高亲和力 Ca1 位点和低亲和力 Ca2 位点。 Ca1 位点在各种枯草杆菌蛋白酶样蛋白酶中是保守的，对于稳定性很重要。该位点在枯草杆菌蛋白酶原 E 中不形成，因为枯草杆菌蛋白酶结构域的 N 末端区域在自动加工时的结构重排对于该位点的形成是必要的。结果，枯草杆菌蛋白酶原 E 没有完全折叠。相比之下，来自热球菌kodakarensis的Pro-Tk-枯草杆菌蛋白酶完全折叠，因为由于前肽和枯草杆菌蛋白酶结构域之间存在插入序列IS1，它不需要在自动加工时进行结构重排来形成Ca1位点。为了检查枯草杆菌蛋白酶原 E 中是否通过在前肽和枯草杆菌蛋白酶结构域之间插入 IS1 来形成 Ca1 位点，构建并表征了具有此插入的枯草杆菌蛋白酶原 E 突变体 IS1-Pro-枯草杆菌蛋白酶 E 及其活性位点突变体 IS1-Pro-S221A 和 IS1-Pro-S221C。 IS1-Pro-S221A 的晶体结构表明该蛋白质已完全折叠并形成 Ca1 位点。在此结构中，IS1 充当连接子，将枯草杆菌蛋白酶结构域的 N 末端带到 Ca1 位点附近。钙结合形式的 IS1-Pro-S221A 比不含钙形式的 IS1-Pro-S221A 稳定 13.1 °C。 IS1-Pro-S221C 的自动处理速度比 Pro-S221C 更快。这些结果表明，IS1 促进 Ca1 位点的形成和枯草杆菌蛋白酶原 E 的完全折叠，从而加速其自动加工。
Subtilisin E is activated from its inactive precursor Pro-subtilisin E by autoprocessing and degradation of the propeptide. Subtilisin E has two calcium binding sites, the high-affinity Ca1 site and the low-affinity Ca2 site. The Ca1 site is conserved in various subtilisin-like proteases and is important for stability. This site is not formed in Pro-subtilisin E, because the structural rearrangement of the N-terminal region of the subtilisin domain upon autoprocessing is necessary for the formation of this site. As a result, Pro-subtilisin E is not fully folded. In contrast, Pro-Tk-subtilisin fromThermococcus kodakarensisis fully folded, because it does not require the structural rearrangement upon autoprocessing for the formation of the Ca1 site due to the presence of the insertion sequence IS1 between the propeptide and subtilisin domains. To examine whether the Ca1 site is formed in Pro-subtilisin E by inserting IS1 between the propeptide and subtilisin domains, the Pro-subtilisin E mutant with this insertion, IS1-Pro-subtilisin E, and its active site mutants, IS1-Pro-S221A and IS1-Pro-S221C, were constructed and characterized. The crystal structure of IS1-Pro-S221A revealed that this protein is fully folded and the Ca1 site is formed. In this structure, IS1 serves as a linker that brings the N-terminus of the subtilisin domain near the Ca1 site. IS1-Pro-S221A in a calcium-bound form was more stable than that in a calcium-free form by 13.1 °C. IS1-Pro-S221C was more rapidly autoprocessed than Pro-S221C. These results suggest that IS1 facilitates the formation of the Ca1 site and the complete folding of Pro-subtilisin E and thereby accelerates its autoprocessing.