6PAK

Insight into subtilisin E-S7 cleavage pattern based on crystal structure and hydrolysates peptide analysis

6PAK の概要

• エントリーDOI: 10.2210/pdb6pak/pdb
• 分子名称: Subtilisin E, CALCIUM ION, 1,2-ETHANEDIOL, ... (4 entities in total)
• 機能のキーワード: subtilisin, hydrolase
• 由来する生物種: Bacillus subtilis (strain 168)
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 58600.76

構造登録者

Tang, H.,Shi, K.,Aihara, H. (登録日: 2019-06-11, 公開日: 2019-10-23, 最終更新日: 2023-10-11)

主引用文献

Tang, H.,Shi, K.,Shi, C.,Aihara, H.,Zhang, J.,Du, G.
Enhancing subtilisin thermostability through a modified normalized B-factor analysis and loop-grafting strategy.
J.Biol.Chem., 294:18398-18407, 2019

PubMed Abstract: Rational design-guided improvement of protein thermostability typically requires identification of residues or regions contributing to instability and introduction of mutations into these residues or regions. One popular method, B-FIT, utilizes B-factors to identify unstable residues or regions and combines them with other strategies, such as directed evolution. Here, we performed structure-based engineering to improve the thermostability of the subtilisin E-S7 (SES7) peptidase. The B-value of each residue was redefined in a normalized B-factor calculation, which was implemented with a refined bioinformatics analysis strategy to identify the critical area (loop 158-162) related to flexibility and to screen for suitable thermostable motif sequences in the Protein Data Bank that can act as transplant loops. In total, we analyzed 445 structures and identified 29 thermostable motifs as candidates. Using these motifs as a starting point, we performed iterative homologous modeling to obtain a desirable chimera loop and introduced five different mutations into this loop to construct thermostable SES7 proteins. Differential scanning fluorimetry revealed increases of 7.3 °C in the melting temperature of an SES7 variant designated M5 compared with the WT. The X-ray crystallographic structure of this variant was resolved at 1.96 Å resolution. The crystal structure disclosed that M5 forms more hydrogen bonds than the WT protein, consistent with design and molecular dynamics simulation results. In summary, the modified B-FIT strategy reported here has yielded a subtilisin variant with improved thermostability and promising industrial applications, supporting the notion that this modified method is a powerful tool for protein engineering.

PubMed: 31615894
DOI: 10.1074/jbc.RA119.010658

実験手法

X-RAY DIFFRACTION (1.98 Å)