PP2C磷酸酶基于flap无规则卷曲选择性识别底物的研究

Metal-dependent protein phosphatases (PP2Cs) are widely distributed in life system. PP2Cs participate in many signal transduction pathways, such as initiation of cancer and plant adaption of stress condition. The solved crystal structures of PP2Cs show the catalytic centers of PP2Cs are highly conserved. It means PP2Cs use the same catalytic mechanism to dephosphorylate substrate. However, many results showed PP2C only can dephosphorylate specific protein that indicates PP2C has special recognition site to bind to substrate. By aligning primary structure and 3-D structure of PP2C, a flexible loop name flap was found to be the candidate of PP2C substrate recognition site, since the primary structure and 3-Dstructure of flap are extremely un-conserved. In this project, applicant will make site-directed mutation of the amino acids of flap and exchange flaps between different PP2Cs. After that, the enzyme kinetics parameters of PP2C mutants and chimeric PP2C towards different substrates (including pNPP and phospho-peptides) will be determined. One or two crystal structures of PP2C or chimeric PP2C will also be solved in this research. The answer of how PP2Cs recognize substrate will help people design specific inhibitor of PP2Cs.

依赖金属离子的蛋白质磷酸酶（PP2C）广泛的存在于生命系统中。PP2C参与了许多重要的信号转导过程，比如癌症的发生及植物适应胁迫环境都与PP2C有关。已解析出来的PP2C晶体结构显示PP2C的催化中心是高度保守的，这说明该类酶利用一致的催化机制对蛋白质进行去磷酸化。尽管如此，许多研究报道显示每种PP2C只对特定的底物去磷酸化，这说明PP2C有帮助其特异性识别底物的部位。通过分析发现PP2C催化中心附近有一段无规则卷曲（命名为flap），所有PP2C flap的一级结构及三级结构都极度不保守，这提示flap利用其独有的结构帮助PP2C特异性识别底物。本项目申请准备对flap区域的氨基酸进行点突变及交换不同PP2C的flap，然后检测所有蛋白的酶动力学特点，还将对PP2C进行晶体结构解析，最后揭示flap如何帮助PP2C特异性识别底物。最终结果将有助于人们开发PP2C特异性抑制剂。

PP2C磷酸酶在生命系统中广泛存在。它们参与了许多重要的信号转导过程，例如癌症的发生以及植物适应胁迫的环境都与PP2C有关。已经解析出来的PP2C的晶体结构显示PP2C具有高度保守的催化中心，这个结构表明此类酶利用一致的催化机制对蛋白质进行去磷酸化。尽管如此，许多的研究结果表明每种PP2C只对特定的底物去磷酸化，这说明它们有帮助其特异性识别底物的部位。通过对晶体结构的分析发现PP2C的催化中心附近有一段无规则卷曲的结构-flap。所有flap的一级结构及三级结构都不保守，这提示flap利用其独有的结构帮助PP2C特异性识别底物。本项目对flap区域的氨基酸进行点突变及交换不同PP2C的flap，检测了所有蛋白的酶动力学特点，对PP2C的晶体结构进行了解析，揭示了flap如何帮助PP2C特异性识别底物。这些研究结果对于有助于开发PP2C特异性抑制剂。