Novel phosphorylation‐dependent regulation in an unstructured protein

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.