Ju Tong, Niu Wei, Guo Jiantao
Department of Chemistry, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
Department of Chemical & Biomolecular Engineering, University of Nebraska-Lincoln , Lincoln, Nebraska 68588, United States.
ACS Chem Biol. 2016 Sep 16;11(9):2551-7. doi: 10.1021/acschembio.6b00555. Epub 2016 Jul 21.
Protein tyrosine O-sulfation is considered as the most common type of post-translational tyrosine modification in nature and plays important roles in extracellular biomolecular interactions. To facilitate the mapping, biological study, and medicinal application of this type of post-translational modification, we seek to evolve a small protein scaffold that recognizes sulfotyrosine with high affinity. We focused our efforts on the engineering of the Src Homology 2 (SH2) domain, which represents the largest class of known phosphotyrosine-recognition domain in nature and has a highly evolvable binding pocket. By using phage display, we successfully engineered the SH2 domain to recognize sulfotyrosine with high affinity. The best mutant, SH2-60.1, displayed more than 1700 fold higher sulfotyrosine-binding affinity than that of the wild-type SH2 domain. We also demonstrated that the evolved SH2 domain mutants could be used to detect sulfoprotein levels on the cell surface. These evolved SH2 domain mutants can be potentially applied to the study of protein tyrosine O-sulfation with proper experimental designs.
蛋白质酪氨酸O-硫酸化被认为是自然界中最常见的翻译后酪氨酸修饰类型，在细胞外生物分子相互作用中发挥着重要作用。为了便于对这种翻译后修饰进行图谱绘制、生物学研究和医学应用，我们试图进化出一种能高亲和力识别硫酸化酪氨酸的小蛋白支架。我们将工作重点放在Src同源2（SH2）结构域的工程改造上，该结构域是自然界中已知的最大一类磷酸酪氨酸识别结构域，具有高度可进化的结合口袋。通过噬菌体展示，我们成功地对SH2结构域进行了工程改造，使其能够高亲和力识别硫酸化酪氨酸。最佳突变体SH2-60.1对硫酸化酪氨酸的结合亲和力比野生型SH2结构域高出1700多倍。我们还证明，进化后的SH2结构域突变体可用于检测细胞表面硫酸化蛋白的水平。通过适当的实验设计，这些进化后的SH2结构域突变体可能会应用于蛋白质酪氨酸O-硫酸化的研究。
