title: 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, whereas its overexpression leads to a growth advantage of prostate cancer cells (Zeng, Y., He, Y., Yang, F., Mooney, S. M., Getzenberg, R. H., Orban, J., and Kulkarni, P. (2011) The cancer/testis antigen prostate-associated gene 4 (PAGE4) is a highly intrinsically disordered protein. J. Biol. Chem. 286, 13985-13994). Phosphorylation of PAGE4 at Thr-51 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 Thr-51. The population of transient turn-like structures increases upon phosphorylation in an ∼20-residue acidic region centered on Thr-51. This central region therefore becomes more compact and more negatively charged, with increasing intramolecular contacts to basic sequence motifs near the N and C termini. Although 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 Thr-51, most likely because of masking the effects of the more compact phosphorylated state. Therefore, phosphorylation of PAGE4 leads to conformational shifts in the dynamic ensemble, with large functional consequences. The changes in the structural ensemble induced by posttranslational modifications are similar conceptually to the conformational switching events seen in some marginally stable ("metamorphic") folded proteins in response to mutation or environmental triggers.
authors.affiliation: From the W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland 20850, the Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742.
authors.forename: Yanan
authors.initials: Y
authors.lastname: He
authors.affiliation: From the W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland 20850, the Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742.
authors.forename: Yihong
authors.initials: Y
authors.lastname: Chen
authors.affiliation: the Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287.
authors.forename: Steven M
authors.initials: SM
authors.lastname: Mooney
authors.affiliation: the Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287.
authors.forename: Krithika
authors.initials: K
authors.lastname: Rajagopalan
authors.affiliation: Shakti BioResearch, Woodbridge, Connecticut 06525, and.
authors.forename: Ajay
authors.initials: A
authors.lastname: Bhargava
authors.affiliation: the Department of Physics, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Elizabeth
authors.initials: E
authors.lastname: Sacho
authors.affiliation: the Department of Physics, North Carolina State University, Raleigh, North Carolina 27695.
authors.forename: Keith
authors.initials: K
authors.lastname: Weninger
authors.affiliation: From the W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland 20850.
authors.forename: Philip N
authors.initials: PN
authors.lastname: Bryan
authors.affiliation: the Department of Urology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21287, pkulkar4@ibbr.umd.edu.
authors.forename: Prakash
authors.initials: P
authors.lastname: Kulkarni
authors.affiliation: From the W. M. Keck Laboratory for Structural Biology, University of Maryland Institute for Bioscience and Biotechnology Research, Rockville, Maryland 20850, the Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, jorban@umd.edu.
authors.forename: John
authors.initials: J
authors.lastname: Orban
mesh_terms: D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm / Q000737:chemistry* / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011471:Prostatic Neoplasms / Q000473:pathology*; D011487:Protein Conformation
chemical_list: D000951:Antigens, Neoplasm; C474505:PAGE4 protein, human
keywords: NMR; cancer/testis antigen; conformational change; intrinsically disordered protein; phosphorylation; prostate cancer; prostate-associated gene 4