Chen Yunting, Jakoncic Jean, Parker Kathlyn A, Carpino Nick, Nassar Nicolas
Department of Physiology and Biophysics, Stony Brook University, Stony Brook, New York 11794-8661, USA.
Biochemistry. 2009 Sep 1;48(34):8129-35. doi: 10.1021/bi9008648.
The C-terminal domain of the suppressor of T cell receptor (TCR) signaling 1 and 2 (Sts-1 and -2) proteins has homology to the 2H-phosphatase family of enzymes. The phosphatase activity of the correspondent Sts-1 domain, Sts-1(PGM), is key for its ability to negatively regulate the signaling of membrane-bound receptors including TCR and the epidermal growth factor receptor (EGFR). A nucleophilic histidine, which is transiently phosphorylated during the phosphatase reaction, is essential for the activity. Here, we present the crystal structure of Sts-2(PGM) in the phosphorylated active form and bound to VO(3), which represent structures of an intermediate and of a transition state analogue along the path of the dephosphorylation reaction. In the former structure, the proposed nucleophilic His366 is the only phoshorylated residue and is stabilized by several interactions with conserved basic residues within the active site. In the latter structure, the vanadium atom sits in the middle of a trigonal bipyramid formed by the three oxygen atoms of the VO(3) molecule, atom NE2 of His366, and an apical water molecule W(a). The V-NE2 bond length (2.25 A) suggests that VO(3) is not covalently attached to His366 and that the reaction mechanism is partially associative. The two structures also suggest a role for Glu476 in activating a uniquely positioned water molecule. In both structures, the conformation of the active site is remarkably similar to the one seen in apo-Sts-2(PGM) suggesting that the spatial arrangement of the catalytic residues does not change during the dephosphorylation reaction.
T细胞受体（TCR）信号转导抑制因子1和2（Sts-1和-2）蛋白的C末端结构域与2H-磷酸酶家族的酶具有同源性。相应的Sts-1结构域Sts-1(PGM)的磷酸酶活性是其负向调节包括TCR和表皮生长因子受体（EGFR）在内的膜结合受体信号转导能力的关键。一个在磷酸酶反应过程中发生瞬时磷酸化的亲核组氨酸对该活性至关重要。在此，我们展示了处于磷酸化活性形式并与VO(3)结合的Sts-2(PGM)的晶体结构，它们代表了去磷酸化反应路径中的一个中间体和一个过渡态类似物的结构。在前一种结构中，推测的亲核His366是唯一的磷酸化残基，并通过与活性位点内保守碱性残基的多种相互作用而稳定。在后一种结构中，钒原子位于由VO(3)分子的三个氧原子、His366的原子NE2和一个顶端水分子W(a)形成的三角双锥的中心。V-NE2键长（2.25 Å）表明VO(3)并非共价连接到His366上，且反应机制部分为缔合反应。这两种结构还表明Glu476在激活一个独特定位的水分子中发挥作用。在这两种结构中，活性位点的构象与脱辅基Sts-2(PGM)中的构象非常相似，这表明在去磷酸化反应过程中催化残基的空间排列没有改变。
