This work explores how phosphorylation of an unstructured protein region in inhibitor-2 (I2) regulates protein phosphatase-1 (PP1) enzyme activity using molecular dynamics (MD). Free I2 is largely unstructured; however, when bound to PP1, three segments adopt a stable structure. In particular, an I2 helix (i-helix) blocks the PP1 active site and inhibits phosphatase activity. I2 phosphorylation in the PP1-I2 complex activates phosphatase activity without I2 dissociation. The I2 Thr74 regulatory phosphorylation site is in an unstructured domain in PP1-I2. PP1-I2 MD demonstrated that I2 phosphorylation promotes early steps of PP1-I2 activation in explicit solvent models. Moreover, phosphorylation-dependent activation occurred in PP1-I2 complexes derived from I2 orthologs with diverse sequences from human, yeast, worm, and protozoa. This system allowed exploration of features of the 73-residue unstructured human I2 domain critical for phosphorylation-dependent activation. These studies revealed that components of I2 unstructured domain are strategically positioned for phosphorylation responsiveness including a transient α-helix. There was no evidence that electrostatic interactions of I2 phosphothreonine74 influenced PP1-I2 activation. Instead, phosphorylation altered the conformation of residues around Thr74. Phosphorylation uncurled the distance between I2 residues Glu71 to Tyr76 to promote PP1-I2 activation, whereas reduced distances reduced activation. This I2 residue Glu71 to Tyr76 distance distribution, independently from Thr74 phosphorylation, controls I2 i-helix displacement from the PP1 active site leading to PP1-I2 activation.

中文翻译：

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非结构化蛋白质中的新型磷酸化依赖性调节。

这项工作探索如何利用分子动力学（MD）来抑制抑制剂2（I2）中非结构化蛋白质区域的磷酸化，从而调节蛋白质磷酸酶1（PP1）的酶活性。免费的I2基本上是非结构化的；但是，当绑定到PP1时，三个段采用稳定的结构。特别是，一个I2螺旋（i-螺旋）会阻止PP1活性位点并抑制磷酸酶活性。PP1-I2复合物中的I2磷酸化可激活磷酸酶活性，而无需I2解离。I2 Thr74调节磷酸化位点在PP1-I2的非结构域中。PP1-I2 MD表明，在明确的溶剂模型中，I2磷酸化可促进PP1-I2活化的早期步骤。此外，磷酸化依赖性活化发生在衍生自I2直向同源物的PP1-I2复合物中，其具有来自人，酵母，蠕虫和原生动物的不同序列。该系统允许探索对磷酸化依赖性激活至关重要的73个残基的非结构化人I2结构域的特征。这些研究表明，I2非结构域的成分在战略上定位于磷酸化反应性，包括瞬时α-螺旋。没有证据表明I2磷酸苏氨酸74的静电相互作用会影响PP1-I2的活化。相反，磷酸化改变了Thr74周围残基的构象。磷酸化消除了I2残基Glu71至Tyr76之间的距离，从而促进了PP1-I2的活化，而减小的距离则降低了活化。I2残基Glu71到Tyr76的距离分布独立于Thr74磷酸化，控制了I2 i-螺旋从PP1活性位点的移位，导致PP1-I2活化。