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Insertion loop‐mediated folding propagation governs efficient maturation of hyperthermophilic Tk‐subtilisin at high temperatures
Author(s) -
Uehara Ryo,
Dan Nanako,
Amesaka Hiroshi,
Yoshizawa Takuya,
Koga Yuichi,
Kanaya Shigenori,
Takano Kazufumi,
Matsumura Hiroyoshi,
Tanaka Shunichi
Publication year - 2021
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1002/1873-3468.14028
Subject(s) - thermococcus , subtilisin , chemistry , folding (dsp implementation) , serine protease , serine , protein folding , stereochemistry , crystallography , biochemistry , archaea , protease , enzyme , gene , electrical engineering , engineering
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.