Structural Basis for the Substrate Specificity of Tobacco Etch Virus Protease*

By J. Phan, A. Zdanov, A. Evdokimov, J. Tropea, H. Peters, R. Kapust, Mi Li, A. Wlodawer, D. Waugh. 2002

260 citations recorded by the source corpus.

Because of its stringent sequence specificity, the 3C-type protease from tobacco etch virus (TEV) is frequently used to remove affinity tags from recombinant proteins. It is unclear, however, exactly how TEV protease recognizes its substrates with such high selectivity. The crystal structures of two TEV protease mutants, inactive C151A and autolysis-resistant S219D, have now been solved at 2.2- and 1.8-Å resolution as complexes with a substrate and product peptide, respectively. The enzyme does not appear to have been perturbed by the mutations in either structure, and the modes of binding of the product and substrate are virtually identical. Analysis of the protein-ligand interactions helps to delineate the structural determinants of substrate specificity and provides guidance for reengineering the enzyme to further improve its utility for biotechnological applications.

Subfields: Ecology, Molecular Biology, Plant Science

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In Molecular Biology

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Citations in the corpus

• Intracellular Detection and Evolution of Site-Specific Proteases Using a Genetic Selection System (2012, background): “Substrate recognition segments of TEV-Pr span three relatively unstructured loops, which are adequately insulated from the bulk of the enzyme [39].”
• Engineering of TEV protease variants by yeast ER sequestration screening (YESS) of combinatorial libraries (2013, background): “Although we did not have direct evidence of the specificity of these two evolved variants at residues flanking P1 in the substrate, we speculate that the TEV-PE10 and TEV-PE21 possess similar specificity to the TEV-P because of the lack of amino acid substitutions near the P1′, P3, and P6 binding su”
• Simultaneous selection and counter‐selection for the directed evolution of proteases in E. coli using a cytoplasmic anchoring strategy (2016, methodology): “The crystal structure of peptide-bound TEVp was used to select residues for mutation (Phan et al., 2002).”
• Enhancing the stability and solubility of TEV protease using in silico design (2007, methodology): “We used the TEV structure complexed with its peptide substrate, with PDB code 1LVM (Phan et al. 2002).”