A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.     

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.     

A systems wide mass spectrometric based linear motif screen to identify dominant in-vivo interacting proteins for the ubiquitin ligase MDM2

Linear motifs mediate protein–protein interactions (PPI) that allow expansion of a target protein interactome at a systems level. This study uses a proteomics approach and linear motif sub-stratifications to expand on PPIs of MDM2. MDM2 is a multi-functional protein with over one hundred known binding partners not stratified by hierarchy or function. A new linear motif based on a MDM2 interaction consensus is used to select novel MDM2 interactors based on Nutlin-3 responsiveness in a cell-based proteomics screen. MDM2 binds a subset of peptide motifs corresponding to real proteins with a range of allosteric responses to MDM2 ligands. We validate cyclophilin B as a novel protein with a consensus MDM2 binding motif that is stabilised by Nutlin-3 in vivo, thus identifying one of the few known interactors of MDM2 that is stabilised by Nutlin-3. These data invoke two modes of peptide binding at the MDM2 N-terminus that rely on a consensus core motif to control the equilibrium between MDM2 binding proteins. This approach stratifies MDM2 interacting proteins based on the linear motif feature and provides a new biomarker assay to define clinically relevant Nutlin-3 responsive MDM2 interactors.     

Structures of low molecular weight inhibitors bound to MDMX and MDM2 reveal new approaches for p53-MDMX/MDM2 antagonist drug discovery

Intensive anticancer drug discovery efforts have been made to develop small molecule inhibitors of the p53-MDM2 and p53-MDMX interactions. We present here the structures of the most potent inhibitors bound to MDM2 and MDMX that are based on the new imidazo-indole scaffold. In addition, the structure of the recently reported spiro-oxindole inhibitor bound to MDM2 is described. The structures indicate how the substituents of a small molecule that bind to the three subpockets of the MDM2/X-p53 interaction should be optimized for effective binding to MDM2 and/or MDMX. While the spiro-oxindole inhibitor triggers significant ligand-induced changes in MDM2, the imidazo-indoles share similar binding modes for MDMX and MDM2, but cause only minimal induced-fit changes in the structures of both proteins. Our study includes the first structure of the complex between MDMX and a small molecule and should aid in developing efficient scaffolds for binding to MDMX and/or MDM2.     

On the interaction mechanisms of a p53 peptide and nutlin with the MDM2 and MDMX proteins: A Brownian dynamics study

The interaction of p53 with its regulators MDM2 and MDMX plays a major role in regulating the cell cycle. Inhibition of this interaction has become an important therapeutic strategy in oncology. Although MDM2 and MDMX share a very high degree of sequence/structural similarity, the small-molecule inhibitor nutlin appears to be an efficient inhibitor only of the p53-MDM2 interaction. Here, we investigate the mechanism of interaction of nutlin with these two proteins and contrast it with that of p53 using Brownian dynamics simulations. In contrast to earlier attempts to examine the bound states of the partners, here we locate initial reaction events in these interactions by identifying the regions of space around MDM2/MDMX, where p53/nutlin experience associative encounters with prolonged residence times relative to that in bulk solution. We find that the initial interaction of p53 with MDM2 is long-lived relative to nutlin, but, unlike nutlin, it takes place at the N- and C termini of the MDM2 protein, away from the binding site, suggestive of an allosteric mechanism of action. In contrast, nutlin initially interacts with MDM2 directly at the clefts of the binding site. The interaction of nutlin with MDMX, however, is very short-lived compared with MDM2 and does not show such direct initial interactions with the binding site. Comparison of the topology of the electrostatic potentials of MDM2 and MDMX and the locations of the initial encounters with p53/nutlin in tandem with structure-based sequence alignment revealed that the origin of the diminished activity of nutlin toward MDMX relative to MDM2 may stem partly from the differing topologies of the electrostatic potentials of the two proteins. Glu25 and Lys51 residues underpin these topological differences and appear to collectively play a key role in channelling nutlin directly toward the binding site on the MDM2 surface and are absent in MDMX. The results, therefore, provide new insight into the mechanism of p53/nutlin interactions with MDM2 and MDMX and could potentially have a broader impact on anticancer drug optimization strategies.     

MDM2 inhibitor Nutlin-3a suppresses proliferation and promotes apoptosis in osteosarcoma cells

Restoring p53 activity by inhibiting the interaction between p53 and the mouse double minutes clone 2 (MDM2) offers an attractive approach to cancer therapy. Nutlin-3a is a small-molecule inhibitor that inhibits MDM2 binding to p53 and subsequent p53-dependent DNA damage signaling. In this study, we determined the efficacy of Nutlin-3a in inducing p53-mediated cell death in osteosarcoma (OS) cell lines both in vivo and in vitro. Targeted disruption of the p53-MDM2 interaction by Nutlin-3a stabilizes p53 and selectively activates the p53 pathway only in OS cells with wild-type p53, resulting in a pronounced anti-proliferative and cytotoxic effect due to G1 cell cycle arrest and apoptosis both in vitro and in vivo. p53 dependence of these alternative outcomes of Nutlin-3a treatment was shown by the abrogation of these effects when p53 was knocked-down by small interfering RNA. These data suggest that the disruption of p53-MDM2 interaction by Nutlin-3a might be beneficial for OS patients with MDM2 amplification and wt p53 status.     

Nutlin-3 protects kidney cells during cisplatin therapy by suppressing Bax/Bak activation

Nutlins, the newly developed small molecule antagonists of MDM2, activate p53 and induce apoptosis in cancer cells, offering a novel strategy of chemotherapy. Recent studies have further suggested synergistic effects of nutlins with other chemotherapeutic drugs. However, it is unclear whether nutlins increase or decrease the side effects of these drugs in normal non-malignant cells or tissues. Cisplatin is a widely used chemotherapy drug, which has a major side effect of kidney injury. Here we show that Nutlin-3 protected kidney cells against cisplatin-induced apoptosis. The cytoprotective effects of Nutlin-3 were not related to its regulation of p53 or consequent gene expression during cisplatin treatment. Moreover, the protective effects were shown in MDM2-, MDM4-, or p53-deficient cells. On the other hand, Nutlin-3 suppressed mitochondrial events of apoptosis during cisplatin incubation, including Bax activation and cytochrome c release. Nutlin-3 attenuated cisplatin-induced oligomerization of Bax and Bak but not their interactions with Bcl-XL. In isolated mitochondria, Nutlin-3 inhibited cytochrome c release induced by Ca2+, Bim peptide, and recombinant tBid. Importantly, it blocked both Bax and Bak oligomerization under these conditions. Together, the results have uncovered a new pharmacological function of nutlins, i.e. suppression of Bax and Bak, two critical mediators of apoptosis.     

Design,synthesis and biological evaluation of novel 3,4,5-trisubstituted aminothiophenes as inhibitors of p53–MDM2 interaction. Part 1

A series of 3,4,5-trisubstituted aminothiophenes were designed, synthesized, and evaluated for their p53–MDM2 binding inhibitory potency and anti-proliferation activities against A549 and PC3 tumor cell lines. Fourteen compounds had appreciably improved MDM2 binding affinities than lead compound MCL0527 (3) and a few compounds showed comparable activities to that of Nutlin-3. Meanwhile, most of the 3,4,5-trisubstituted aminothiophenes displayed better or equivalent anti-proliferation activities against wild-type p53 cell line A549 compared to that of Nutlin-3. Over ten compounds exhibited desirable selective profiles of p53 status. Particularly, compounds 9, 16 and 18 displayed 22-, 6- and 22-fold selectivity of p53 status, respectively, much better than that of Nutlin-3 (fourfold).     

Structure of Human MDM4 N-Terminal Domain Bound to a Single-Domain Antibody

The N-terminal domain of MDM4 binds to the N-terminal transactivation domain of the tumor suppressor p53 and is an important negative regulator of its transactivation activity. As such, inhibition of the binding of MDM4 to p53 is a target for anticancer therapy. The protein has not been crystallized satisfactorily for structural studies without the addition of an N-terminal p53 peptide. We selected a single-domain antibody (VH9) that bound to the human domain with a dissociation constant of 44 nM. We solved the structure of the complex at 2.0-Å resolution. The asymmetric unit contained eight molecules of VH9 and four molecules of MDM4. A molecule of VH9 was located in each transactivation domain binding site, and the four non-MDM4-bound VH9 domains provided additional crystal contacts. There are differences between the structures of human MDM4 domain bound to VH9 and those of human and zebra fish MDM4 bound to a p53 peptide. Molecular dynamics simulations showed that the binding pocket in the three MDM4 structures converged to a common conformation after removal of the ligands, indicating that the differences are due to induced fit. The largest conformational changes were for the MDM4 molecules bound to p53. The simulated and observed structures should aid rational drug design. The use of single-domain antibodies to aid crystallization by creating a molecular scaffold may have a wider range of applications.     

An shRNA barcode screen provides insight into cancer cell vulnerability to MDM2 inhibitors

The identification of the cellular targets of small molecules with anticancer activity is crucial to their further development as drug candidates. Here, we present the application of a large-scale RNA interference-based short hairpin RNA (shRNA) barcode screen to gain insight in the mechanism of action of nutlin-3 (1). Nutlin-3 is a small-molecule inhibitor of MDM2, which can activate the p53 pathway. Nutlin-3 shows strong antitumor effects in mice, with surprisingly few side effects on normal tissues. Aside from p53, we here identify 53BP1 as a critical mediator of nutlin-3-induced cytotoxicity. 53BP1 is part of a signaling network induced by DNA damage that is frequently activated in cancer but not in healthy tissues. Our results suggest that nutlin-3's tumor specificity may result from its ability to turn a cancer cell-specific property (activated DNA damage signaling) into a weakness that can be exploited therapeutically.     

Disruption of the MDM2�Cp53 interaction strongly potentiates p53-dependent apoptosis in cisplatin-resistant human testicular carcinoma cells via the Fas/FasL pathway

Wild-type p53 has a major role in the response and execution of apoptosis after chemotherapy in many cancers. Although high levels of wild-type p53 and hardly any TP53 mutations are found in testicular cancer (TC), chemotherapy resistance is still observed in a significant subgroup of TC patients. In the present study, we demonstrate that p53 resides in a complex with MDM2 at higher cisplatin concentrations in cisplatin-resistant human TC cells compared with cisplatin-sensitive TC cells. Inhibition of the MDM2–p53 interaction using either Nutlin-3 or MDM2 RNA interference resulted in hyperactivation of the p53 pathway and a strong induction of apoptosis in cisplatin-sensitive and -resistant TC cells. Suppression of wild-type p53 induced resistance to Nutlin-3 in TC cells, demonstrating the key role of p53 for Nutlin-3 sensitivity. More specifically, our results indicate that p53-dependent induction of Fas membrane expression (∼threefold) and enhanced Fas/FasL interactions at the cell surface are important mechanisms of Nutlin-3-induced apoptosis in TC cells. Importantly, an analogous Fas-dependent mechanism of apoptosis upon Nutlin-3 treatment is executed in wild-type p53 expressing Hodgkin lymphoma and acute myeloid leukaemia cell lines. Finally, we demonstrate that Nutlin-3 strongly augmented cisplatin-induced apoptosis and cell kill via the Fas death receptor pathway. This effect is most pronounced in cisplatin-resistant TC cells.     

Nutlin-3 induces apoptosis,disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G₁ cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of “KS-like” tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. These results suggest that Nutlin-3 might serve as a novel therapy for KS.     

Functional consequences of retro-inverso isomerization of a miniature protein inhibitor of the p53–MDM2 interaction

Peptide retro-inverso isomerization is thought to be functionally neutral and has been widely used as a tool for designing proteolytically stable d-isomers to recapitulate biological activities of their parent l-peptides. Despite success in a wide range of applications, exceptions amply exist that clearly defy this rule of thumb when parent l-peptides adopt an α-helical conformation in their bound state. The detrimental energetic effect of retro-inverso isomerization of an α-helical l-peptide on its target protein binding has been estimated to be 3.0–3.4 kcal/mol. To better understand how the retro-inverso isomer of a structured protein works at the molecular level, we chemically synthesized and functionally characterized the retro-inverso isomer of a rationally designed miniature protein termed stingin of 18 amino acid residues, which adopts an N-terminal loop and a C-terminal α-helix stabilized by two intra-molecular disulfide bridges. Stingin emulated the transactivation peptide of the p53 tumor suppressor protein and bound with high affinity and via its C-terminal α-helix to MDM2 and MDMX—the two negative regulators of p53. We also prepared the retro isomer and d-enantiomer of stingin for comparative functional studies using fluorescence polarization and surface plasmon resonance techniques. We found that retro-inverso isomerization of l-stingin weakened its MDM2 binding by 720 fold (3.9 kcal/mol); while enantiomerization of l-stingin drastically reduced its binding to MDM2 by three orders of magnitude, sequence reversal completely abolished it. Our findings demonstrate the limitation of peptide retro-inverso isomerization in molecular mimicry and reinforce the notion that the strategy works poorly with biologically active α-helical peptides due to inherent differences at the secondary and tertiary structural levels between an l-peptide and its retro-inverso isomer despite their similar side chain topologies at the primary structural level.¹     

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. Proteins 2013; 81:1686–1698. © 2013 Wiley Periodicals, Inc.     

An allosteric-feedback mechanism for protein-assisted group I intron splicing

The I-AniI maturase facilitates self-splicing of a mitochondrial group I intron in Aspergillus nidulans. Binding occurs in at least two steps: first, a specific but labile encounter complex rapidly forms and then this intermediate is slowly resolved into a native, catalytically active RNA/protein complex. Here we probe the structure of the RNA throughout the assembly pathway. Although inherently unstable, the intron core, when bound by I-AniI, undergoes rapid folding to a near-native state in the encounter complex. The next transition includes the slow destabilization and docking into the core of the peripheral stacked helix that contains the 5' splice site. Mutational analyses confirm that both transitions are important for native complex formation. We propose that protein-driven destabilization and docking of the peripheral stacked helix lead to subtle changes in the I-AniI binding site that facilitate native complex formation. These results support an allosteric-feedback mechanism of RNA-protein recognition in which proteins engaged in an intermediate complex can influence RNA structure far from their binding sites. The linkage of these changes to stable binding ensures that the protein and RNA do not get sequestered in nonfunctional complexes.     

Molecular Dynamics Calculations of Wild Type vs. Mutant Protein C: Relationship Between Binding Affinity to Endothelial Cell Protein C Receptor and Hereditary Disease

Abstract

Molecular dynamics simulations of the protein C γ-carboxyglutamic acid (Gla) domain and endothelial cell protein C receptor (EPCR) complex were performed to determine the effect of a hereditary disease, which results in a mutation (Gla 25 → Lys) in the protein C Gla domain. Our results suggest that the Gla 25 → Lys mutation causes a significant reduction in the binding force between protein C Gla domain and EPCR due to destabilization of the helix structure of EPCR and displacement of a Ca²⁺ ion.     

Restriction endonuclease EcoRI alters the enantiomeric preference of chiral metallointercalators for DNA: an illustration of a protein-induced DNA conformational change

A conformational change in the DNA plasmid ColE1 appears to occur upon specific binding of the restriction endonuclease EcoRI. Enzyme association alters the chiral discrimination found in binding metallointercalators to DNA sites. The complexes tris(1,10-phenanthroline)ruthenium(II), Ru(phen)3(2+), tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II), Ru(DIP)3(2+), and tris(4,7-diphenyl-1,10-phenanthroline)cobalt(III), Co(DIP)3(3+), in general, bind stereoselectively to DNA helices, with enantiomers possessing the delta configuration bound preferentially by right-handed B-DNA. In the presence of EcoRI, however, this enantioselectivity is altered. The chiral intercalators, at micromolar concentrations, inhibit the reaction of EcoRI, but for each enantiomeric pair it is the lambda enantiomer, which binds only poorly to a B-DNA helix, that inhibits EcoRI preferentially. Kinetic studies in the presence of lambda-Ru(DIP)3(2+) indicate that the enzyme inhibition occurs as a result of the lambda enantiomer binding to the enzyme-DNA complex as well as to the free enzyme. Furthermore, photolytic strand cleavage experiments using Co(DIP)3(3+) indicate that the metal complex interacts directly at the protein-bound DNA site. Increasing concentrations of bound EcoRI stimulate photoactivated cleavage of the DNA helix by lambda-Co(DIP)3(3+), until a protein concentration is reached where specific DNA recognition sites are saturated with enzyme. Thus, although lambda-Co(DIP)3(3+) does not bind closely to the DNA in the absence of enzyme, specific binding of EcoRI appears to alter the DNA structure so as to permit the close association of the lambda isomer to the DNA helix. Mapping experiments demonstrate that this association leads to photocleavage of DNA by the cobalt complex at or very close to the EcoRI recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)