Nunn Christine M, Jeeves Mark, Cliff Matthew J, Urquhart Gillian T, George Roger R, Chao Luke H, Tscuchia Yugo, Djordjevic Snezana
Department of Biochemistry and Molecular Biology, University College London, Gower Street, London, WC1E 6BT, UK.
J Mol Biol. 2005 Jul 1;350(1):145-55. doi: 10.1016/j.jmb.2005.04.013.
Tobacco etch virus (TEV) protease is a cysteine protease exhibiting stringent sequence specificity. The enzyme is widely used in biotechnology for the removal of the affinity tags from recombinant fusion proteins. Crystal structures of two TEV protease mutants as complexes with a substrate and a product peptide provided the first insight into the mechanism of substrate specificity of this enzyme. We now report a 2.7A crystal structure of a full-length inactive C151A mutant protein crystallised in the absence of peptide. The structure reveals the C terminus of the protease bound to the active site. In addition, we determined dissociation constants of TEV protease substrate and product peptides using isothermal titration calorimetry for various forms of this enzyme. Data suggest that TEV protease could be inhibited by the peptide product of autolysis. Separate modes of recognition for native substrates and the site of TEV protease self-cleavage are proposed.
烟草蚀纹病毒（TEV）蛋白酶是一种具有严格序列特异性的半胱氨酸蛋白酶。该酶在生物技术领域被广泛用于从重组融合蛋白中去除亲和标签。两种TEV蛋白酶突变体与底物和产物肽形成复合物的晶体结构首次揭示了这种酶的底物特异性机制。我们现在报道在无肽情况下结晶的全长无活性C151A突变蛋白的2.7埃晶体结构。该结构揭示了蛋白酶的C末端与活性位点结合。此外，我们使用等温滴定量热法测定了TEV蛋白酶底物和产物肽对于该酶各种形式的解离常数。数据表明TEV蛋白酶可能被自溶的肽产物抑制。本文提出了对天然底物的不同识别模式以及TEV蛋白酶自我切割的位点。
