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

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.

Keywords

Protein engineering / C-Terminal modification / Cysteine protease domain / Self-cleavage tag / AlphaFold2 / Biological Sciences / Biochemistry and Cell Biology

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RIGHTS & PERMISSIONS

Jilin University, The Editorial Department of Chemical Research in Chinese Universities and Springer-Verlag GmbH