P1′ specificity of the S219V/R203G mutant tobacco etch virus protease

• Published in: Proteins, structure, function, and bioinformatics Vol. 92; no. 9; pp. 1085 - 1096
• Main Authors: Golda, Mária, Hoffka, Gyula, Cherry, Scott, Tropea, Joseph E., Lountos, George T., Waugh, David S., Wlodawer, Alexander, Tőzsér, József, Mótyán, János András
• Format: Journal Article
• Language: English
• Published: Hoboken, USA John Wiley & Sons, Inc 01-09-2024; Wiley Subscription Services, Inc
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Amino acids; Binding sites; Catalytic Domain; Cleavage; Controlled conditions; Cysteine proteinase; Endopeptidases - chemistry; Endopeptidases - genetics; Endopeptidases - metabolism; Enzymes; enzymology; Fusion protein; fusion tag removal; Gene Expression; Inactivation; Kinetics; molecular dynamics; Mutation; Nuclear fusion; Potyvirus - chemistry; Potyvirus - enzymology; Potyvirus - genetics; Protease; protein structure; Proteins; Proteolysis; Reagents; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Resistant mutant; Stability; Substrate Specificity; Substrates; Tags; TEV protease; Tobacco; tobacco etch virus; fusion tag removal; protein structure; tobacco etch virus; molecular dynamics; TEV protease; enzymology
• ISSN: 0887-3585; 1097-0134; 1097-0134
• DOI: 10.1002/prot.26693

Proteases that recognize linear amino acid sequences with high specificity became indispensable tools of recombinant protein technology for the removal of various fusion tags. Due to its stringent sequence specificity, the catalytic domain of the nuclear inclusion cysteine protease of tobacco etch virus (TEV PR) is also a widely applied reagent for enzymatic removal of fusion tags. For this reason, efforts have been made to improve its stability and modify its specificity. For example, P1′ autoproteolytic cleavage‐resistant mutant (S219V) TEV PR was found not only to be nearly impervious to self‐inactivation, but also exhibited greater stability and catalytic efficiency than the wild‐type enzyme. An R203G substitution has been reported to further relax the P1′ specificity of the enzyme, however, these results were obtained from crude intracellular assays. Until now, there has been no rigorous comparison of the P1′ specificity of the S219V and S219V/R203G mutants in vitro, under carefully controlled conditions. Here, we compare the P1′ amino acid preferences of these single and double TEV PR mutants. The in vitro analysis was performed by using recombinant protein substrates representing 20 P1′ variants of the consensus TENLYFQ*SGT cleavage site, and synthetic oligopeptide substrates were also applied to study a limited set of the most preferred variants. In addition, the enzyme–substrate interactions were analyzed in silico. The results indicate highly similar P1′ preferences for both enzymes, many side‐chains can be accommodated by the S1′ binding sites, but the kinetic assays revealed lower catalytic efficiency for the S219V/R203G than for the S219V mutant.