Insertion loop-mediated folding propagation governs efficient maturation of hyperthermophilic Tk-subtilisin at high temperatures

FEBS Letters

article

Ryo Uehara¹  Nanako Dan¹  Hiroshi Amesaka⁴  Takuya Yoshizawa¹  Yuichi Koga⁵  Shigenori Kanaya⁵  Kazufumi Takano⁴  Hiroyoshi Matsumura¹  Shun-ichi Tanaka¹ 

[1] Department of Biotechnology, College of Life Sciences, Ritsumeikan University;Ritsumeikan Global Innovation Research Organization, Ritsumeikan University;Division of Cancer Cell Regulation, Aichi Cancer Center Research Institute;Department of Biomolecular Chemistry, Kyoto Prefectural University;Department of Material and Life Science, Graduate School of Engineering, Osaka University

关键词: archaea;    folding;    hyperthermophile;    maturation;    subtilase;   

DOI  :  10.1002/1873-3468.14028

来源: John Wiley & Sons Ltd.

【 摘 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.

【 授权许可】

Unknown