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

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.

MutL traps MutS at a DNA mismatch.

DNA mismatch repair (MMR) identifies and corrects errors made during replication. In all organisms except those expressing MutH, interactions between a DNA mismatch, MutS, MutL, and the replication processivity factor (ß-clamp or PCNA) activate the latent MutL endonuclease to nick the error-containing daughter strand. This nick provides an entry point for downstream repair proteins. Despite the well-established significance of strand-specific nicking in MMR, the mechanism(s) by which MutS and MutL assemble on mismatch DNA to allow the subsequent activation of MutL's endonuclease activity by ß-clamp/PCNA remains elusive. In both prokaryotes and eukaryotes, MutS homologs undergo conformational changes to a mobile clamp state that can move away from the mismatch. However, the function of this MutS mobile clamp is unknown. Furthermore, whether the interaction with MutL leads to a mobile MutS-MutL complex or a mismatch-localized complex is hotly debated. We used single molecule FRET to determine that Thermus aquaticus MutL traps MutS at a DNA mismatch after recognition but before its conversion to a sliding clamp. Rather than a clamp, a conformationally dynamic protein assembly typically containing more MutL than MutS is formed at the mismatch. This complex provides a local marker where interaction with ß-clamp/PCNA could distinguish parent/daughter strand identity. Our finding that MutL fundamentally changes MutS actions following mismatch detection reframes current thinking on MMR signaling processes critical for genomic stability.

Cancer/testis antigen PAGE4, a regulator of c-Jun transactivation, is phosphorylated by homeodomain-interacting protein kinase 1, a component of the stress-response pathway.

Prostate-associated gene 4 (PAGE4) is a cancer/testis antigen that is typically restricted to the testicular germ cells but is aberrantly expressed in cancer. Furthermore, PAGE4 is developmentally regulated with dynamic expression patterns in the developing prostate and is also a stress-response protein that is upregulated in response to cellular stress. PAGE4 interacts with c-Jun, which is activated by the stress-response kinase JNK1, and plays an important role in the development and pathology of the prostate gland. Here, we have identified homeodomain-interacting protein kinase 1 (HIPK1), also a component of the stress-response pathway, as a kinase that phosphorylates PAGE4 at T51. We show that phosphorylation of PAGE4 is critical for its transcriptional activity since mutating this T residue abolishes its ability to potentiate c-Jun transactivation. In vitro single molecule FRET indicates phosphorylation results in compaction of (still) intrinsically disordered PAGE4. Interestingly, however, while our previous observations indicated that the wild-type nonphosphorylated PAGE4 protein interacted with c-Jun [Rajagopalan , K. et al. ( 2014 ) Biochim, Biophys. Acta 1842 , 154 -163], here we show that phosphorylation of PAGE4 weakens its interaction with c-Jun in vitro. These data suggest that phosphorylation induces conformational changes in natively disordered PAGE4 resulting in its decreased affinity for c-Jun to promote interaction of c-Jun with another, unidentified, partner. Alternatively, phosphorylated PAGE4 may induce transcription of a novel partner, which then potentiates c-Jun transactivation. Regardless, the present results clearly implicate PAGE4 as a component of the stress-response pathway and uncover a novel link between components of this pathway and prostatic development and disease.

The Stress-response protein prostate-associated gene 4, interacts with c-Jun and potentiates its transactivation.

The Cancer/Testis Antigen (CTA), Prostate-associated Gene 4 (PAGE4), is a stress-response protein that is upregulated in prostate cancer (PCa) especially in precursor lesions that result from inflammatory stress. In cells under stress, translocation of PAGE4 to mitochondria increases while production of reactive oxygen species decreases. Furthermore, PAGE4 is also upregulated in human fetal prostate, underscoring its potential role in development. However, the proteins that interact with PAGE4 and the mechanisms underlying its pleiotropic functions in prostatic development and disease remain unknown. Here, we identified c-Jun as a PAGE4 interacting partner. We show that both PAGE4 and c-Jun are overexpressed in the human fetal prostate; and in cell-based assays, PAGE4 robustly potentiates c-Jun transactivation. Single-molecule Förster resonance energy transfer experiments indicate that upon binding to c-Jun, PAGE4 undergoes conformational changes. However, no interaction is observed in presence of BSA or unilamellar vesicles containing the mitochondrial inner membrane diphosphatidylglycerol lipid marker cardiolipin. Together, our data indicate that PAGE4 specifically interacts with c-Jun and that, conformational dynamics may account for its observed pleiotropic functions. To our knowledge, this is the first report demonstrating crosstalk between a CTA and a proto-oncogene. Disrupting PAGE4/c-Jun interactions using small molecules may represent a novel therapeutic strategy for PCa.

DNA repair factor MRE11/RAD50 cleaves 3'-phosphotyrosyl bonds and resects DNA to repair damage caused by topoisomerase 1 poisons.

MRE11-RAD50 is a highly conserved multifunctional DNA repair factor. Here, we show that MRE11-RAD50 cleaves the covalent 3'-phosphotyrosyl-DNA bonds that join topoisomerase 1 (Top1) to the DNA backbone and that are the hallmark of damage caused by Top1 poisons such as camptothecin. Cleavage generates a 3'-phosphate DNA end that MRE11-RAD50 can resect in an ATP-regulated reaction, to produce a 3'-hydroxyl that can prime repair synthesis. The 3'-phosphotyrosyl cleavage activity maps to the MRE11 active site. These results define a new activity of MRE11 and distinguish MRE11-RAD50 functions in repair of Top1-DNA complexes and double-strand breaks.

A model for oligomeric regulation of APOBEC3G cytosine deaminase-dependent restriction of HIV.

APOBEC3G (A3G) restricts HIV-1 infection by catalyzing processive C --> U deaminations on single-stranded DNA (ssDNA) with marked 3' --> 5' deamination polarity. Here we show that A3G exists in oligomeric states whose composition is dictated primarily by interactions with DNA, with salt playing an important, yet secondary, role. Directional deaminations correlate with the presence of dimers, tetramers, and larger oligomers observed by atomic force microscopy, and random deaminations appear to correlate mainly with monomers. The presence of a 30-nt weakly deaminated "dead" zone located at the 3'-ssDNA end implies the presence of a preferred asymmetric direction for A3G catalysis. Single turnover reaction rates reveal a salt-dependent inhibition of C deamination toward the 3'-ssDNA region, offering a molecular basis underlying A3G deamination polarity. Presteady state analysis demonstrates rapid diffusion-limited A3G-ssDNA binding, a slower salt-dependent conformational change, possibly indicative of DNA wrapping, and long (5-15 min) protein-DNA complex lifetimes. We suggest that diverse A3G oligomerization modes contribute to the human immunodeficiency virus, type 1, proviral DNA mutational bias.

Direct visualization of asymmetric adenine-nucleotide-induced conformational changes in MutL alpha.

MutL alpha, the heterodimeric eukaryotic MutL homolog, is required for DNA mismatch repair (MMR) in vivo. It has been suggested that conformational changes, modulated by adenine nucleotides, mediate the interactions of MutL alpha with other proteins in the MMR pathway, coordinating the recognition of DNA mismatches by MutS alpha and the activation of MutL alpha with the downstream events that lead to repair. Thus far, the only evidence for these conformational changes has come from X-ray crystallography of isolated domains, indirect biochemical analyses, and comparison to other members of the GHL ATPase family to which MutL alpha belongs. Using atomic force microscopy (AFM), coupled with biochemical techniques, we demonstrate that adenine nucleotides induce large asymmetric conformational changes in full-length yeast and human MutL alpha and that these changes are associated with significant increases in secondary structure. These data reveal an ATPase cycle in which sequential nucleotide binding, hydrolysis, and release modulate the conformational states of MutL alpha.

Activation-induced deaminase, AID, is catalytically active as a monomer on single-stranded DNA.

Hypermutation and class switch recombination of immunoglobulin genes are antigen-activated mechanisms triggered by AID, a cytidine deaminase. AID deaminates cytidine residues in the DNA of the variable and the switch regions of the immunoglobulin locus. The resulting uracil induces error-prone DNA synthesis in the case of hypermutation or DNA breaks that activate non-homologous recombination in the case of class switch recombination. In vitro studies have demonstrated that AID deaminates single-stranded but not double-stranded substrates unless AID is in a complex with RPA and the substrate is actively undergoing transcription. However, it is not clear whether AID deaminates its substrates primarily as a monomer or as a higher order oligomer. To examine the oligomerization state of AID alone and in the presence of single-stranded DNA substrates of various structures, including loops embedded in double-stranded DNA, we used atomic force microscopy (AFM) to visualize AID protein alone or in complex with DNA. Surprisingly, AFM results indicate that most AID molecules exist as a monomer and that it binds single-stranded DNA substrates as a monomer at concentrations where efficient deamination of single-stranded DNA substrates occur. The rate of deamination, under conditions of excess and limiting protein, also imply that AID can deaminate single-stranded substrates as a monomer. These results imply that non-phosphorylated AID is catalytically active as a monomer on single-stranded DNA in vitro, including single-stranded DNA found in loops similar to those transiently formed in the immunoglobulin switch regions during transcription.