Insertion loop‐mediated folding propagation governs efficient maturation of hyperthermophilic Tk‐subtilisin at high temperatures
Insertion loop‐mediated folding propagation governs efficient maturation of hyperthermophilic Tk‐subtilisin at high temperatures
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DOI：
10.1002/1873-3468.14028
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发表时间：
2020-12
期刊：
影响因子：
3.5
通讯作者：
R. Uehara;Nanako Dan;Hiroshi Amesaka;T. Yoshizawa;Y. Koga;S. Kanaya;K. Takano;H. Matsumura;Shun-ichi Tanaka
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文献类型：
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作者：
R. Uehara;Nanako Dan;Hiroshi Amesaka;T. Yoshizawa;Y. Koga;S. Kanaya;K. Takano;H. Matsumura;Shun-ichi Tanaka
与嗜温对应物相比，来自超嗜热古菌 Thermococcus kodakarensis 的丝氨酸蛋白酶 Tk-枯草杆菌蛋白酶在其表面具有三个插入环 (IS1-IS3)。尽管 IS1 和 IS2 是 Tk-枯草杆菌蛋白酶在高温下成熟所必需的，但 IS3 的作用仍然未知。 CD光谱显示，IS3缺失将Tk-枯草杆菌蛋白酶折叠阻止在中间状态，其中形成中心核，但随后的折叠传播到末端子结构域并未发生。晶体学分析证明，IS3 中天冬氨酸残基的丙氨酸取代扰乱了环内氢键网络，导致高温下折叠受损。考虑到 IS3 在超嗜热同系物中的高度保守性，我们认为 IS3 的存在对于超嗜热枯草杆菌蛋白酶在高温环境中的折叠非常重要。
The serine protease Tk‐subtilisin from the hyperthermophilic archaeon Thermococcus kodakarensis possesses three insertion loops (IS1‐IS3) on its surface, as compared to its mesophilic counterparts. Although IS1 and IS2 are required for maturation of Tk‐subtilisin at high temperatures, the role of IS3 remains unknown. Here, CD spectroscopy revealed that IS3 deletion arrested Tk‐subtilisin folding at an intermediate state, in which the central nucleus was formed, but the subsequent folding propagation into terminal subdomains did not occur. Alanine substitution of the aspartate residue in IS3 disturbed the intraloop hydrogen‐bonding network, as evidenced by crystallographic analysis, resulting in compromised folding at high temperatures. Taking into account the high conservation of IS3 across hyperthermophilic homologues, we propose that the presence of IS3 is important for folding of hyperthermophilic subtilisins in high‐temperature environments.