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.