Strategic Amino Acid Mutations in CPD Cleavage Motif: Impacts on Hydrolysis and C-Terminal Modification Efficiency

doi:10.1007/s40242-025-5013-0

Chemical Research in Chinese Universities ›› 2025, Vol. 41 ›› Issue (3): 592-600.doi: 10.1007/s40242-025-5013-0

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Strategic Amino Acid Mutations in CPD Cleavage Motif: Impacts on Hydrolysis and C-Terminal Modification Efficiency

Ansor YASHINOV^1,2, ZOU Xiangman^1,6, HANG Jiayin^1,2, LIU Zhi^4,5, SONG Fengnan³, ZENG Yue¹, YANG Yang³, XIA Fei¹, TANG Feng¹, SHI Wei¹, HUANG Wei^1,2,7

1. State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P. R. China;
2. University of Chinese Academy of Sciences, Beijing 100049, P. R. China;
3. School of Pharmaceutical Science and Technology, Hangzhou Institute of Advanced Study, Hangzhou 310024, P. R. China;
4. Lingang Laboratory, Shanghai 200031, P. R. China;
5. School of Physical Science and Technology, Shanghai Tech University, Shanghai 201210, P. R. China;
6. Key Laboratory of Structure-based Drug Design and Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, P. R. China;
7. Shanghai GlycanLink Biotech. Co., Ltd., Shanghai 201210, P. R. China

Received:2025-01-21 Revised:2025-03-27 Online:2025-06-01 Published:2025-05-27
Contact: HUANG Wei,E-mail:huangwei@simm.ac.cn;SHI Wei,E-mail:shiwei1@simm.ac.cn E-mail:huangwei@simm.ac.cn;shiwei1@simm.ac.cn
Supported by:
This work was supported by the National Science Fund for Distinguished Young Scholars, China (No. 82325045), the National Natural Science Foundation of China (Nos. 82204183, 22277126), the Shanghai Sail Program, China (No. 22YF1457400), and the Hangzhou Innovation and Entrepreneurship Leading Team Project, China (No. TD2020005).

Abstract

Abstract: Precise modification of the C-terminus of proteins is crucial for investigating protein-protein interaction and enhancing protein functionalities. While traditional methods face challenges due to multiple reactive sites, recent advancements have introduced cysteine protease domain (CPD) tag for efficient C-terminal modifications. CPD, when fused with proteins of interest (POI), can facilitate concurrent hydrolysis and amidation under Inositol hexakisphosphate (InsP6) activation. Herein, we explored the influence of substituting the Ala residue following Leu in the CPD cleavage motif (VDALADGK) with each of the 19 other amino acids. By creating a series of green fluorescent protein (GFP)-CPD fusion constructs, we evaluated their hydrolysis and amidation efficiencies. Our results revealed that mutations to Ser and Asn significantly enhanced C-terminal modification, while Pro substitution completely hindered hydrolysis activity. Additionally, we demonstrated the successful labeling of a Ser mutant with a fluorescent probe, establishing its potential for Förster resonance energy transfer (FRET) applications. Structural analyses using AlphaFold2 indicated that the observed variations in activity could be attributed to the differences in molecular interactions and the flexibility of the substituted amino acids. Overall, this research highlights the utility of strategically designed mutations in enhancing C-terminal modifications, offering valuable insights for future protein engineering endeavors.

Key words: Protein engineering, C-Terminal modification, Cysteine protease domain, Self-cleavage tag, AlphaFold2

Ansor YASHINOV, ZOU Xiangman, HANG Jiayin, LIU Zhi, SONG Fengnan, ZENG Yue, YANG Yang, XIA Fei, TANG Feng, SHI Wei, HUANG Wei. Strategic Amino Acid Mutations in CPD Cleavage Motif: Impacts on Hydrolysis and C-Terminal Modification Efficiency[J]. Chemical Research in Chinese Universities, 2025, 41(3): 592-600.

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Strategic Amino Acid Mutations in CPD Cleavage Motif: Impacts on Hydrolysis and C-Terminal Modification Efficiency

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