P53 Transactivation Domain Research Articles

Interaction between Transactivation Domain of p53 and Middle Part of TBP-Like Protein (TLP) Is Involved in TLP-Stimulated and p53-Activated Transcription from the p21 Upstream Promoter

TBP-like protein (TLP) is involved in transcriptional activation of an upstream promoter of the human p21 gene. TLP binds to p53 and facilitates p53-activated transcription from the upstream promoter. In this study, we clarified that in vitro affinity between TLP and p53 is about one-third of that between TBP and p53. Extensive mutation analyses revealed that the TLP-stimulated function resides in transcription activating domain 1 (TAD1) in the N-terminus of p53. Among the mutants, #22.23, which has two amino acid substitutions in TAD1, exhibited a typical mutant phenotype. Moreover, #22.23 exhibited the strongest mutant phenotype for TLP-binding ability. It is thus thought that TLP-stimulated and p53-dependent transcriptional activation is involved in TAD1 binding of TLP. #22.23 had a decreased transcriptional activation function, especially for the upstream promoter of the endogenous p21 gene, compared with wild-type p53. This mutant did not facilitate p53-dependent growth repression and etoposide-mediated cell-death as wild-type p53 does. Moreover, mutation analysis revealed that middle part of TLP, which is requited for p53 binding, is involved in TLP-stimulated and p53-dependent promoter activation and cell growth repression. These results suggest that activation of the p21 upstream promoter is mediated by interaction between specific regions of TLP and p53.

Structural basis for the conserved binding mechanism of MDM2-inhibiting peptides and anti-apoptotic Bcl-2 family proteins

The interaction between tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins serves a critical role in the transcription-independent apoptosis mechanism of p53. Our previous studies showed that an MDM2-inhibiting motif (residues 15–29) in the p53 transactivation domain (p53TAD) mediates the interaction with anti-apoptotic Bcl-2 family proteins. In this study, we provided structural models of the complexes between the MDM2-inhibiting p53TAD peptide and Mcl-1, Bcl-w, and Kaposi sarcoma-associated herpes virus (KSHV) Bcl-2 using NMR chemical shift perturbation data. The binding mode of the MDM2-inhibiting p53TAD peptide is highly conserved among the anti-apoptotic Bcl-2 family proteins despite their distinct specificities for pro-apoptotic Bcl-2 family proteins. We also identified the binding of a phage-display-derived MDM2-inhibiting peptide 12-1 to anti-apoptotic Bcl-XL protein by using NMR spectroscopy. The structural model of the Bcl-XL/12-1 peptide complex revealed that the conserved residues Phe4, Trp8, and Leu11 in the MDM2-inhibiting peptide fit into a hydrophobic cleft of Bcl-XL in a manner similar to that of pro-apoptotic Bcl-2 homology 3 (BH3) peptides. Our results shed light on the mechanism underlying dual-targeting of the FxxxWxxL-based α-helical motif to MDM2 and anti-apoptotic Bcl-2 family proteins for anticancer therapy.

Abstract P3-03-05: Modulation of the metastatic propensity of triple-negative breast cancer cells harboring p53 mutations

Abstract The mutation of p53 is implicated in the increased metastatic potential of triple-negative (ER—/PR—/Her2—) breast cancer (TNBC). The low survival rates of advanced metastatic cancer make it critical not only to understand the mechanisms underlying the functional implications of p53 mutations, but also to identify ways to regulate their impact in cancer cell physiology. A common gain-of-function activity of the mutant p53 protein is the enhancement of cell migration and invasion. We have found that a hydrocarbon-stapled p53 transactivation domain peptide (SAH-p53) designed to disrupt the interaction with p53 negative regulators HDM2 and HDMX also has the ability to inhibit the interaction between mutant p53 (mtp53) and HDM2/HDMX. In addition, SAH-p53 was found to inhibit the migration and invasion of MDA-MB-231 cells (mtp53), while exhibiting no alteration in their proliferative ability. The capacity of SAH-p53 to inhibit MDA-MB-231 cell migration was not altered in the absence of HDMX, suggesting an alternative mechanism of action for SAH-p53. The propensity of MDA-MB-231 cells to migrate and invade through Matrigel in response to transforming growth factor b (TGF-β), epidermal growth factor (EGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) was decreased by treatment with SAH-p53. Furthermore, increased concentrations of these growth factors displayed a synergistic effect with SAH-p53 in the inhibition of cell migration, suggesting cross-talk between receptor signaling and SAH-p53 activity. This effect was accompanied by an alteration in stress fiber formation and dissolution of focal adhesions. While the knockdown of mutant p53 in MDA-MB-231 cells significantly decreased migration on its own, inhibition of migration by SAH-p53 was not seen in the EGFR/p53-null MDA-MB-157 cells, suggesting a need for either the p53 or EGFR protein for the inhibitory activity of SAH-p53. Moreover, a SAH-p53-induced decrease in the total levels of EGFR and integrin beta1, together with alterations in actin stability, suggests a defect in receptor recycling, a process required for cell migration known to be modulated by an interaction between mutant p53 and p63. These data show that the use of the SAH-p53 peptide provides a novel avenue to study the molecular interactions that drive metastatic behavior originated by p53 gain-of-function mutations. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-03-05.

Targeting of p53 peptide analogues to anti-apoptotic Bcl-2 family proteins as revealed by NMR spectroscopy

Inhibition of the interaction between the p53 tumor suppressor and its negative regulator MDM2 is of great importance to cancer therapy. The anti-apoptotic Bcl-2 family proteins are also attractive anti-cancer molecular targets, as they are key regulators of apoptotic cell death. Previously, we reported the interactions between the p53 transactivation domain (p53TAD) and diverse members of the anti-apoptotic Bcl-2 family proteins. In this study, we investigated the binding of MDM2-inhibiting p53TAD peptide analogues, p53-MDM2/MDMX inhibitor (PMI) and pDI, with anti-apoptotic Bcl-2 family proteins, Bcl-XL and Bcl-2, by using NMR spectroscopy. The NMR chemical shift perturbation data demonstrated the direct binding of the p53 peptide analogues to Bcl-XL and Bcl-2 and showed that the PMI and pDI peptides bind to a conserved hydrophobic groove of the anti-apoptotic Bcl-2 family proteins. Furthermore, the structural model of the Bcl-XL/PMI peptide complex showed that the binding mode of the PMI peptide is highly similar to that of pro-apoptotic Bcl-2 homology 3 (BH3) peptides. Finally, our structural comparison provided a molecular basis for how the same PMI peptide can bind to two distinct anti-cancer target proteins Bcl-XL and MDM2, which may have potential applications for multi-targeting cancer therapy.

Abstract 19038: Epigenetic Regulation of Canonical TNFa Pathway by HDAC1 Determines Survival of Cardiac Myocytes

Gene transcription is regulated by post-translation modifications. Histone deacetylases (HDACs) remove acetyl-groups from histone and non-histone factors inhibiting transcription. Pro-inflammatory cytokines such as TNFα activate the canonical Nuclear Factor -κB (NF-κB) pathway. Earlier we established a cytoprotective role for NF-κB in the heart. Though a causal relationship for HDAC1 and NF-κB has been established, the impact of HDAC1 on TNFα signaling is unknown. Herein, we demonstrate HDAC1 provides a molecular switch for determining cell survival in the TNFα pathway. In contrast to vehicle treated control cells, TNFα treated cells displayed a marked increase in NF-κB gene transcription. Notably, cells treated with TNFα were indistinguishable from vehicle controls cells with respect to viability. Interestingly, HDAC activity was reduced in cells treated with TNFα. Conversely, in the presence of HDAC1, NF-κB gene transcription by TNFα was repressed, resulting in mitochondrial perturbations and widespread cell death. Heterologous fusion proteins comprised of yeast Gal4 DNA binding domain fused in frame to the NF-κB p65 transactivation domain were preferentially repressed by HDAC1. Moreover, transcription mediated by Gal4VP16 protein from herpes virus was unaffected by HDAC1 in cardiac myocytes. Mutations that abrogate known catalytic activities of HDAC1, small interference RNA or pharmacological inhibition of HDAC1, restored NF-κB signaling and suppressed cell death induced by TNFα. These data provide the first evidence for an obligate link between HDAC1 and canonical TNFα pathway for cell survival of cardiac myocytes.

Anti-apoptosis Proteins Mcl-1 and Bcl-xL Have Different p53-Binding Profiles

One of the transcription-independent mechanisms of the tumor suppressor p53 discovered in recent years involves physical interaction between p53 and proteins of the Bcl-2 family. In this paper, significant differences between the interaction of p53 with Mcl-1 and Bcl-xL were demonstrated by NMR spectroscopy and isothermal titration calorimetry. Bcl-xL was found to bind strongly to the p53 DNA-binding domain (DBD) with a dissociation constant (Kd) of ~600 nM, whereas Mcl-1 binds to the p53 DBD weakly with a dissociation constant in the mM range. In contrast, the p53 transactivation domain (TAD) binds weakly to Bcl-xL with a Kd ~ 300-500 μM and strongly to Mcl-1 with a Kd ~ 10-20 μM. NMR titrations indicate that although the p53 TAD binds to the BH3-binding grooves of both Bcl-xL and Mcl-1, Bcl-xL prefers to bind to the first subdomain (TAD1) in the p53 TAD, and Mcl-1 prefers to bind to the second subdomain (TAD2). Therefore, Mcl-1 and Bcl-xL have different p53-binding profiles. This indicates that the detailed interaction mechanisms are different, although both Mcl-1 and Bcl-xL can mediate transcription-independent cytosolic roles of p53. The revealed differences in binding sites and binding affinities should be considered when BH3 mimetics are used in cancer therapy development.

Abstract 268: Epigenetic Regulation Of Canonical Tnfa Pathway By Hdac1 Determines Survival Of Cardiac Myocytes

Gene transcription is regulated by post-translation modifications. Histone deacetylases (HDACs) remove acetyl-groups from histone and non-histone factors inhibiting transcription. Pro-inflammatory cytokines such as TNFα activate the canonical Nuclear Factor -κB (NF-κB) pathway. Earlier we established a cytoprotective role for NF-κB in the heart. Though a causal relationship for HDAC1 and NF-κB has been established, the impact of HDAC1 on TNFα signaling is unknown. Herein, we demonstrate HDAC1 provides a molecular switch for determining cell survival in the TNFα pathway. In contrast to vehicle treated control cells, TNFα treated cells displayed a marked increase in NF-κB gene transcription. Notably, cells treated with TNFα were indistinguishable from vehicle controls cells with respect to viability. Interestingly, HDAC activity was reduced in cells treated with TNFα. Conversely, in the presence of HDAC1, NF-κB gene transcription by TNFα was repressed, resulting in mitochondrial perturbations and widespread cell death. Heterologous fusion proteins comprised of yeast Gal4 DNA binding domain fused in frame to the NF-κB p65 transactivation domain were preferentially repressed by HDAC1. Moreover, transcription mediated by Gal4VP16 protein from herpes virus was unaffected by HDAC1 in cardiac myocytes. Mutations that abrogate known catalytic activities of HDAC1, small interference RNA or pharmacological inhibition of HDAC1, restored NF-κB signaling and suppressed cell death induced by TNFα. These data provide the first evidence for an obligate link between HDAC1 and canonical TNFα pathway for cell survival of cardiac myocytes.

Structural divergence is more extensive than sequence divergence for a family of intrinsically disordered proteins

The p53 transactivation domain (p53TAD) is an intrinsically disordered protein (IDP) domain that undergoes coupled folding and binding when interacting with partner proteins like the E3 ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70. The secondary structure and dynamics of six closely related mammalian homologues of p53TAD were investigated using nuclear magnetic resonance (NMR) spectroscopy. Differences in both transient secondary structure and backbone dynamics were observed for the homologues. Many of these differences were localized to the binding sites for MDM2 and RPA70. The amount of transient helical secondary structure observed for the MDM2 binding site was lower for the dog and mouse homologues, compared with human, and the amount of transient helical secondary structure observed for the RPA70 binding site was higher for guinea pig and rabbit, compared with human. Differences in the amount of transient helical secondary structure observed for the MDM2 binding site were directly related to amino acid substitutions occurring on the solvent exposed side of the amphipathic helix that forms during the p53TAD/MDM2 interaction. Differences in the amount of transient helical secondary structure were not as easily explained for the RPA70 binding site because of its extensive sequence divergence. Clustering analysis shows that the divergence in the transient secondary structure of the p53TAD homologues exceeds the amino acid sequence divergence. In contrast, strong correlations were observed between the backbone dynamics of the homologues and the sequence identity matrix, suggesting that the dynamic behavior of IDPs is a conserved evolutionary feature.

Impact of the K24N mutation on the transactivation domain of p53 and its binding to murine double-minute clone 2.

The level of the p53 transcription factor is negatively regulated by the E3 ubiquitin ligase murine double-minute clone 2 (MDM2). The interaction between p53 and MDM2 is essential for the maintenance of genomic integrity for most eukaryotes. Previous structural studies revealed that MDM2 binds to p53 transactivation domain (p53TAD) from residues 17 to 29. The K24N mutation of p53TAD changes a lysine at position 24 to an asparagine. This mutation occurs naturally in the bovine family and is also found in a rare form of human gestational cancer called choriocarcinoma. In this study, we have investigated how the K24N mutation affects the affinity, structure, and dynamics of p53TAD binding to MDM2. Nuclear magnetic resonance studies of p53TAD show that the K24N mutant is more flexible and has less transient helical secondary structure than the wild type. Isothermal titration calorimetry measurements demonstrate that these changes in structure and dynamics do not significantly change the binding affinity for p53TAD-MDM2. Finally, free-energy perturbation and standard molecular dynamic simulations suggest the negligible affinity change is due to a compensating interaction energy between the K24N mutant and the MDM2 when it is bound. Overall, the data suggest that the K24N-MDM2 complex is able to, at least partly, compensate for an increase in the conformational entropy in unbound K24N with an increase in the bound-state electrostatic interaction energy.

Site-specific NMR mapping and time-resolved monitoring of serine and threonine phosphorylation in reconstituted kinase reactions and mammalian cell extracts

We outline NMR protocols for site-specific mapping and time-resolved monitoring of protein phosphorylation reactions using purified kinases and mammalian cell extracts. These approaches are particularly amenable to intrinsically disordered proteins and unfolded, regulatory protein domains. We present examples for the ¹⁵N isotope-labeled N-terminal transactivation domain of human p53, which is either sequentially reacted with recombinant enzymes or directly added to mammalian cell extracts and phosphorylated by endogenous kinases. Phosphorylation reactions with purified enzymes are set up in minutes, whereas NMR samples in cell extracts are prepared within 1 h. Time-resolved NMR measurements are performed over minutes to hours depending on the activities of the probed kinases. Phosphorylation is quantitatively monitored with consecutive 2D ¹H-¹⁵N band-selective optimized-flip-angle short-transient (SOFAST)-heteronuclear multiple-quantum (HMQC) NMR experiments, which provide atomic-resolution insights into the phosphorylation levels of individual substrate residues and time-dependent changes thereof, thereby offering unique advantages over western blotting and mass spectrometry.

Abstract 3436: Integrated profiling of p53 wild-type cell lines identifies differentially responsive populations and a gene expression signature that predicts sensitivity to SAR405838, a potent and selective disruptor of the p53-MDM2 interaction.

Abstract The tumor suppressor protein p53 functions at the center of a complex biological network that translates diverse stress signals into cell cycle arrest or apoptosis. MDM2 inhibits p53 activity by binding to the transactivation domain of p53 and by acting as a p53-specific E3 ubiquitin ligase. The disruption of the interaction between p53 and MDM2 with small molecular mass molecules has demonstrated encouraging single agent anti-tumor activity in preclinical in vitro and in vivo settings. While p53 wild-type status is necessary for response to antagonists of the p53-MDM2 protein-protein interaction (PPI), there is diversity in response to such therapeutic agents across p53 wild-type tumor models. As such, the ability to predict p53 wild-type tumor responsiveness to antagonists of the p53-MDM2 PPI is an unmet need that could significantly impact clinical development of modulators of the p53-MDM2 PPI. Here, we explored the effect of SAR405838, a selective p53-MDM2 antagonist, on a panel of 65 p53 wild-type cell lines derived from different tumors of origin. Over 80% of these cell lines showed some degree of sensitivity to SAR405838 when used as a single agent. Three broad response classes were observed: non-responders, responders involving apoptosis, and responders not involving apoptosis. A small number of tissue types were consistently enriched among responders (melanoma and leukemia) and non-responders (cervical and uterine tumors). By comparing pre-treatment gene expression profiling data with the single agent sensitivity to SAR405838, we identified a signature of 39 genes that clearly predicts non-response vs response. Within these 39 genes, CDKN2A was upregulated in non-responders, and MDM2 was upregulated in responders. Gene set enrichment analysis and GO category enrichments were consistent with predicted sensitivity in melanoma and leukemia, and indicate that a gene expression signature of loss of p53 pathway function predicts lack of single agent activity of SAR405838. 3-fold cross-validation of this classifier showed an accuracy of 94%, and principle component analyses was used to down-select the signature to the smallest number of genes carrying the maximum information and derive the optimal cut-off for making prospective response predictions. These data indicate that the development of p53-MDM2 PPI modulators could be targeted to a subset of p53 wild-type cancer patients and eliminate more predicted non-responders than those harbouring somatic mutations in p53. Citation Format: James Watters, Steve Rowley, Laurent Debussche. Integrated profiling of p53 wild-type cell lines identifies differentially responsive populations and a gene expression signature that predicts sensitivity to SAR405838, a potent and selective disruptor of the p53-MDM2 interaction. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3436. doi:10.1158/1538-7445.AM2013-3436

Abstract 2223: Identification of novel protein modulators of p53 family function using photochemical crosslinking of stapled peptides.

Abstract Considered to be the “guardian of the genome,” the tumor suppressor p53 works to counteract genetic mutation through cell cycle arrest, DNA repair or apoptosis to prevent proliferation of damaged cells. Dysfunction of the p53 pathway is a large driver of tumorigenesis, with an estimated more than 50% of cancers bearing some p53 abnormality; hence, the normalization of p53 function is considered an important clinical goal for cancer therapy. Many of the interactions involving p53 have been extensively studied; however, absent structural information, the identification of the precise nature of the binding interactions proves challenging, more so if they are transient. Using the hydrocarbon stapled peptide methodology previously used by our group to study interactions mediated by alpha-helices, we have begun identifying novel binding targets of the p53 transactivation domain (TAD) as well as its related proteins p63 and p73 through biochemically specific, covalent photochemical crosslinking reactions. Methodologically, we have developed the use of photoreactive stapled alpha helices (pSAHs) representing a unique way to use highly selective, biochemically specific cross-linking reagents. These can be used more broadly to study protein-protein binding interactions in cell-free and cell based assays. The technique has allowed us to learn more about the biologically active state of proteins that interact with the p53-family member TADs as well as the identification of unexplored binding partners and their mechanism of action in disease. Citation Format: Kenneth M. Felsenstein, Kathryn M. Headley, Kevin A. Murray, Elisabeth A. Russell, Federico Bernal. Identification of novel protein modulators of p53 family function using photochemical crosslinking of stapled peptides. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2013-2223

Epigenetic regulation of canonical TNFα pathway by HDAC1 determines survival of cardiac myocytes

Gene transcription is regulated by post-translation modifications. Histone deacetylases (HDACs) remove acetyl groups from histone and non-histone factors inhibiting transcription. Proinflammatory cytokines such as TNFα activate the canonical nuclear factor-κB (NF-κB) pathway. Earlier we established a cytoprotective role for NF-κB in the heart. Though a causal relationship for HDAC1 and NF-κB has been established, the impact of HDAC1 on TNFα signaling is unknown. Herein, we demonstrate that HDAC1 provides a molecular switch for determining cell survival in the TNFα pathway. In contrast to vehicle-treated control cells, TNFα-treated cells displayed a marked increase in NF-κB gene transcription. Notably, cells treated with TNFα were indistinguishable from vehicle controls cells with respect to viability. Interestingly, HDAC activity was reduced in cells treated with TNFα. Conversely, in the presence of HDAC1, NF-κB gene transcription by TNFα was repressed, resulting in mitochondrial perturbations and widespread cell death. Heterologous fusion proteins comprised of yeast Gal4 DNA binding domain fused in frame to the NF-κB p65 transactivation domain were preferentially repressed by HDAC1. Moreover, transcription mediated by Gal4VP16 protein from herpes virus was unaffected by HDAC1 in cardiac myocytes. Mutations that abrogate known catalytic activities of HDAC1, small interference RNA, or pharmacological inhibition of HDAC1 restored NF-κB signaling and suppressed cell death induced by TNFα. These data provide the first evidence for an obligate link between HDAC1 and canonical TNFα pathway for cell survival of cardiac myocytes.

Global Conformational Selection and Local Induced Fit for the Recognition between Intrinsic Disordered p53 and CBP

The transactivation domain (TAD) of tumor suppressor p53 can bind with the nuclear coactivator binding domain (NCBD) of cyclic-AMP response element binding protein (CBP) and activate transcription. NMR experiments demonstrate that both apo-NCBD and TAD are intrinsic disordered and bound NCBD/TAD undergoes a transition to well folded. The recognition mechanism between intrinsic disordered proteins is still hotly debated. Molecular dynamics (MD) simulations in explicit solvent are used to study the recognition mechanism between intrinsic disordered TAD and NCBD. The average RMSD values between bound and corresponding apo states and Kolmogorov-Smirnov P test analysis indicate that TAD and NCBD may follow an induced fit mechanism. Quantitative analysis indicates there is also a global conformational selection. In summary, the recognition of TAD and NCBD might obey a local induced fit and global conformational selection. These conclusions are further supported by high-temperature unbinding kinetics and room temperature landscape analysis. These methods can be used to study the recognition mechanism of other intrinsic disordered proteins.

Dual-site Interactions of p53 Protein Transactivation Domain with Anti-apoptotic Bcl-2 Family Proteins Reveal a Highly Convergent Mechanism of Divergent p53 Pathways

Molecular interactions between the tumor suppressor p53 and the anti-apoptotic Bcl-2 family proteins play an important role in the transcription-independent apoptosis of p53. The p53 transactivation domain (p53TAD) contains two conserved ΦXXΦΦ motifs (Φ indicates a bulky hydrophobic residue and X is any other residue) referred to as p53TAD1 (residues 15-29) and p53TAD2 (residues 39-57). We previously showed that p53TAD1 can act as a binding motif for anti-apoptotic Bcl-2 family proteins. In this study, we have identified p53TAD2 as a binding motif for anti-apoptotic Bcl-2 family proteins by using NMR spectroscopy, and we calculated the structures of Bcl-X(L)/Bcl-2 in complex with the p53TAD2 peptide. NMR chemical shift perturbation data showed that p53TAD2 peptide binds to diverse members of the anti-apoptotic Bcl-2 family independently of p53TAD1, and the binding between p53TAD2 and p53TAD1 to Bcl-X(L) is competitive. Refined structural models of the Bcl-X(L)·p53TAD2 and Bcl-2·p53TAD2 complexes showed that the binding sites occupied by p53TAD2 in Bcl-X(L) and Bcl-2 overlap well with those occupied by pro-apoptotic BH3 peptides. Taken together with the mutagenesis, isothermal titration calorimetry, and paramagnetic relaxation enhancement data, our structural comparisons provided the structural basis of p53TAD2-mediated interaction with the anti-apoptotic proteins, revealing that Bcl-X(L)/Bcl-2, MDM2, and cAMP-response element-binding protein-binding protein/p300 share highly similar modes of binding to the dual p53TAD motifs, p53TAD1 and p53TAD2. In conclusion, our results suggest that the dual-site interaction of p53TAD is a highly conserved mechanism underlying target protein binding in the transcription-dependent and transcription-independent apoptotic pathways of p53.

Atomic Structure of Dual-Specificity Phosphatase 26, a Novel p53 Phosphatase

Regulation of p53 phosphorylation is critical to control its stability and biological activity. Dual-specificity phosphatase 26 (DUSP26) is a brain phosphatase highly overexpressed in neuroblastoma, which has been implicated in dephosphorylating phospho-Ser20 and phospho-Ser37 in the p53 transactivation domain. In this paper, we report the 1.68 Å crystal structure of a catalytically inactive mutant (Cys152Ser) of DUSP26 lacking the first 60 N-terminal residues (ΔN60-C/S-DUSP26). This structure reveals the architecture of a dual-specificity phosphatase domain related in structure to Vaccinia virus VH1. DUSP26 adopts a closed conformation of the protein tyrosine phosphatase (PTP)-binding loop, which results in an unusually shallow active site pocket and buried catalytic cysteine. A water molecule trapped inside the PTP-binding loop makes close contacts both with main chain and with side chain atoms. The hydrodynamic radius (R(H)) of ΔN60-C/S-DUSP26 measured from velocity sedimentation analysis (R(H) ∼ 22.7 Å) and gel filtration chromatography (R(H) ∼ 21.0 Å) is consistent with an ∼18 kDa globular monomeric protein. Instead in crystal, ΔN60-C/S-DUSP26 is more elongated (R(H) ∼ 37.9 Å), likely because of the extended conformation of C-terminal helix α9, which swings away from the phosphatase core to generate a highly basic surface. As in the case of phosphatase MKP-4, we propose that a substrate-induced conformational change, possibly involving rearrangement of helix α9 with respect to the phosphatase core, allows DUSP26 to adopt a catalytically active conformation. The structural characterization of DUSP26 presented in this paper provides the first atomic insight into this disease-associated phosphatase.

Structural Divergence Exceeds Sequence Divergence for a Family of Intrinsically Disordered Proteins

Several sequence based evolutionary analyses have concluded that many intrinsically disordered proteins and disordered regions evolve faster than ordered proteins. However, there are only a couple of studies that demonstrate the physical consequences of this rapid evolution. The transactivation domain of the tumor suppressor protein p53 (p53TAD) is an intrinsically disordered protein with some transient helical structure that is stabilized by binding to partners like the E3 ubiquitin ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70. The secondary structure and backbone dynamics of five mammalian orthologues of p53TAD were investigated using nuclear magnetic resonance (NMR) spectroscopy. Significant differences in secondary structure and dynamics were observed in the binding sites of MDM2 and RPA70 between the orthologues. The degree of transient helical structure observed in the MDM2 binding site was directly related to amino acid substitutions occurring on the solvent exposed side of the amphipathic helix that forms in the bound state, with dog, cow, and mouse orthologues having the lowest fraction of transient helical structure and the fastest backbone dynamics. Differences were also observed in the RPA70 binding site with the guinea pig and rabbit orthologues showing significantly higher propensity for transient helical structure than the other orthologues. Clustering analysis shows greater divergence in secondary structure than expected based on sequence divergence. However, strong correlations were found between the backbone dynamics and the sequence identity of the orthologues. This result is consistent with a previous study from our group and suggests that the dynamic behavior of IDPs is under positive selection.

Adeno-associated Virus-mediated, Mifepristone-regulated Transgene Expression in the Brain

Gene therapy, in its current configuration, is irreversible and does not allow control over transgene expression in case of side effects. Only few regulated vector systems are available, and none of these has reached clinical applicability yet. The mifepristone (Mfp)-regulated Gene Switch (GS) system is characterized by promising features such as being composed of mainly human components and an approved small-molecule drug as an inducer. However, it has not yet been evaluated in adeno-associated virus (AAV) vectors, neither has it been tested for applicability in viral vectors in the central nervous system (CNS). Here, we demonstrate that the GS system can be used successfully in AAV vectors in the brain, and that short-term induced glial cell line-derived neurotrophic factor (GDNF) expression prevented neurodegeneration in a rodent model of Parkinson's disease (PD). We also demonstrate repeated responsiveness to the inducer Mfp and absence of immunological tissue reactions in the rat brain. Human equivalent dosages of Mfp used in this study were lower than those used safely for treatment of psychiatric threats, indicating that the inducer could be safely applied in patients. Our results suggest that the GS system in AAV vectors is well suited for further development towards clinical applicability.

De Novo Generation of Bound Structures for an Intrinsically Disordered Protein using Only Alpha Carbon Chemical Shifts

Intrinsically disordered proteins (IDPs) challenge our traditional notions of protein structure/function relationships because they are highly dynamic in their native state and do not form tertiary structures. To design new drugs for IDPs it is essential to generate realistic structural ensembles for these proteins. We have recently developed a method called broad ensemble generation with re-weighting (BEGR) where a million or more diverse IDP structures are generated and then re-weighted to fit experimental data. Results show that bound state structures of the disordered p53 transactivation domain (p53TAD) are reproducibly generated, even though the BEGR method is using experimental data for unbound p53TAD. These bound state structures were found for wild type p53TAD and a mutant that increases the transient helical secondary structure of one of the binding sites using only the alpha carbon chemical shift data.

A small molecule directly inhibits the p53 transactivation domain from binding to replication protein A

Replication protein A (RPA), essential for DNA replication, repair and DNA damage signalling, possesses six ssDNA-binding domains (DBDs), including DBD-F on the N-terminus of the largest subunit, RPA70. This domain functions as a binding site for p53 and other DNA damage and repair proteins that contain amphipathic alpha helical domains. Here, we demonstrate direct binding of both ssDNA and the transactivation domain 2 of p53 (p53TAD2) to DBD-F, as well as DBD-F-directed dsDNA strand separation by RPA, all of which are inhibited by fumaropimaric acid (FPA). FPA binds directly to RPA, resulting in a conformational shift as determined through quenching of intrinsic tryptophan fluorescence in full length RPA. Structural analogues of FPA provide insight on chemical properties that are required for inhibition. Finally, we confirm the inability of RPA possessing R41E and R43E mutations to bind to p53, destabilize dsDNA and quench tryptophan fluorescence by FPA, suggesting that protein binding, DNA modulation and inhibitor binding all occur within the same site on DBD-F. The disruption of p53–RPA interactions by FPA may disturb the regulatory functions of p53 and RPA, thereby inhibiting cellular pathways that control the cell cycle and maintain the integrity of the human genome.