Structure and Function of Metal-Dependent Protein Phosphatases

我们已经结晶并解决了人类PPM1A的催化结构域与环状磷酸肽抑制剂络合的结构，该抑制剂模拟了PPM1A的生理底物之一p38 MAPK的激活环(1)。因此，我们已经证实在后生动物PPM磷酸酶的活性部位怀疑存在第三种金属离子。进一步的生物物理和计算研究表明，通过降低Flap子结构域的构象灵活性，复合物形成导致更紧凑的溶液构象。我们还观察到底物在活性位点的位置允许溶剂进入不稳定的第三个金属结合位点。磷酸肽底物的酶动力学研究证明了一个随机顺序的双底物机制，结合的底物与不稳定的金属离子之间存在大量相互作用。我们的工作揭示了调节PPM磷酸酶活性的固有机制的结构和热力学基础。

We have crystallized and solved the structure of the catalytic domain of human PPM1A complexed with a cyclic phosphopeptide inhibitor that mimics the activation loop of p38 MAPK (1), one of the physiological substrates of PPM1A. We therefore have confirmed the suspected presence of a third metal ion in the active site of metazoan PPM phosphatases. Further biophysical and computational studies suggested that complex formation results in a more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetic studies with a phosphopeptide substrate provided evidence for a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. Our work has revealed the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases. 1. Debnath et al. (2018) A trapped human PPM1A-phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity. J. Biol. Chem. 293, 7993-8008.