Phosphorylation-Induced Conformational Ensemble Switching in an Intrinsically Disordered Cancer/Testis Antigen

Abstract Prostate-associated Gene-4 (PAGE4) is an intrinsically disordered Cancer/Testis Antigen that is up-regulated in the fetal and diseased human prostate. Knocking down PAGE4 expression results in cell death while its over-expression leads to a growth advantage of prostate cancer cells [Zeng et al. (2011) J. Biol. Chem. 286, 13985-13994]. Phosphorylation of PAGE4 at T51 is critical for potentiating c-Jun transactivation, an important factor in controlling cell growth, apoptosis, and stress response. Using NMR spectroscopy, we show that the PAGE4 polypeptide chain has local and long-range conformational preferences that are perturbed by site-specific phosphorylation at T51. The population of transient turn-like structures increases upon phosphorylation in an approximately 20-residue acidic region centered on T51. This central region thus becomes more compact and more negatively charged, with increasing intramolecular contacts to basic sequence motifs near the N- and C-termini. While flexibility is decreased in the central region of phospho-PAGE4, the polypeptide chain remains highly dynamic overall. PAGE4 utilizes a transient helical structure adjacent to the central acidic region to bind c-Jun with low affinity in vitro. The binding interaction is attenuated by phosphorylation at T51, most likely due to masking effects of the more compact phosphorylated state. Thus, phosphorylation of PAGE4 leads to conformational shifts in the dynamic ensemble with large functional consequences. The changes in the structural ensemble induced by post-translational modifications are similar conceptually to the conformational switching events seen in some marginally stable (metamorphic) folded proteins in response to mutation or environmental triggers.