CRYSTAL STRUCTURE OF A YERSINIA TYROSINE PHOSPHATASE

蛋白质酪氨酸残基的磷酸化是细胞 传播膜受体信号和调节 细胞生长和致癌转化。 最近发现的一种 蛋白质酪氨酸磷酸酶的不断增长的家族， 特异性地从磷酸酪氨酸中除去磷酸部分， 含有蛋白质，是启动这些信号通路所必需的 并用于控制细胞生长的水平。 尽管中心 在这些调节过程中，没有任何已知的三级结构， 的关键酶。 本提案介绍了初步结果， 确定细菌X射线晶体结构的策略 蛋白酪氨酸磷酸酶，有和没有结合的抑制剂， 底物，这将作为一个范例，了解酶的 功能和特异性。 致病菌耶尔森氏菌是导致一系列人类和 从痢疾到黑死病的啮齿动物疾病。 一个基本 其毒力的决定因素是分泌的蛋白酪氨酸磷酸酶 名为YOP 51。 Yop 51可能是一种可以进入宿主细胞的毒素， 干扰免疫细胞磷酸化水平和活化 途径，并允许耶尔森菌破坏宿主的免疫系统 监视 这种酶的催化部分是高度同源的 与人酪氨酸磷酸酶具有相似的底物特异性， 都有一个相同的催化机制， 半胱氨酸。 此外，它可以大量表达为 生物化学和结构研究。 已经获得了大的，衍射良好的脱辅基酶晶体。 已经收集了数据，正在寻找重原子衍生物。 中求进工作总 磷酸酶的衍射晶体与 还获得了有效的含氧阴离子抑制剂钨酸盐，其不同于 当地人 这种结构可能模仿独特的磷酸半胱氨酸酶 过渡态 此外，还描述了 使与酶结合的磷酸酪氨酸肽底物结晶 不能进行底物周转的变体。 这将揭示酶是如何 催化磷酸盐去除，为什么它是磷酸酪氨酸特异性的， 底物-蛋白质相互作用如何决定酶的特异性。

Phosphorylation of protein tyrosine residues is a cell's primary mechanism for propagating membrane receptor signals and for regulating cell growth and oncogenic transformation. A recently identified and growing family of protein tyrosine phosphatases, enzymes that specifically remove the phosphate moieties from phosphotyrosine- containing proteins, are essential for priming these signalling pathways and for controlling the levels of cell growth. Despite the centrality of these regulatory processes, no tertiary structures are known of any of the key enzymes. This proposal describes preliminary results and strategies to determine the X-ray crystal structure of a bacterial protein tyrosine phosphatase, with and without bound inhibitors and substrates, which will serve as a paradigm for understanding the enzyme's function and specificity. The pathogenic bacterium Yersinia is responsible for a range of human and rodent diseases from diarrhea to the bubonic plague. An essential determinant of its virulence is a secreted protein tyrosine phosphatase termed Yop51. Yop51 is likely a toxin which may enter host cells, interfere with immune cell phosphorylation levels and activation pathways, and allow Yersinia to subvert the host's immune system surveillance. The catalytic portion of this enzyme is highly homologous to human tyrosine phosphatases, has similar substrate specificity, and has an identical catalytic mechanism centered around an essential cysteine. Additionally, it can be expressed in large amounts for biochemical and structural studies. Large, well-diffracting crystals have been obtained of the apoenzyme. Data have been collected and a search for heavy atom derivatives is in progress. Diffracting crystals of the phosphatase complexed with the potent oxyanion inhibitor tungstate were also obtained and differ from the native. This structure may mimick the unique phosphocysteine enzyme transition state. In addition, procedures are described for crystallizing a phosphotyrosine peptide substrate bound to an enzyme variant incapable of substrate turnover. This will reveal how the enzyme catalyzes phosphate removal, why it is specific for phosphotyrosine, and how substrate-protein interactions define the enzyme's specificity.