Low Energy Conformations for S100 Binding Peptide from the Negative Regulatory Domain of p53

We have computed the low energy conformations of the negative regulatory domain of p53, residues 374–388 using Empirical Conformational Energies of Peptides Program including solvation and computed the statistical weights of distinct conformational states. We find that there are two high probability conformations, one an α-helix from Lys 374–Lys 381, followed by another helical structure involving Lys 382–Glu 388 (statistical weight of 0.48) and an all-α-helix for the entire sequence (statistical weight of 0.23). Both structure are superimposable on the NMR structure of this sequence bound to the S100 protein. The global minimum structure (statistical weight of 0.014) is a beta structure from Gly 374–Arg 379 followed by an α-helix from His 380–Glu 388. Based on these results, we propose a possible strategy for enhancement of p53 anti-tumor activity in cancer cells. Since the structure of this sequence bound to the sirtuin protein, Sir2, is a β-sheet, we further propose that the global minimum may be an intermediate on the α-β structure transition.     

Myelin P0 Glycoprotein and a Synthetic Peptide Containing the Palmitoylation Site Are Both Autoacylated

Abstract: P₀, the major protein of the PNS myelin, is palmitoylated at the cytoplasmic Cys¹⁵³. To gain insights into the mechanism of P₀ acylation, the in vitro palmitoylation of both P₀ and a synthetic Cys¹⁵³-containing octapeptide was studied. Incubation of PNS myelin membranes or isolated P₀ with [³H]palmitoyl-CoA resulted in specific labeling of this protein, suggesting that the reaction is nonenzymatic. Incorporation of the labeled fatty acid into P₀ was not affected by boiling the isolated P₀ for 15 min before incubation or by adding sciatic nerve homogenate to the reaction mixture, which confirms the nonezymatic nature of the reaction. After chemical deacylation, P₀ was palmitoylated at a higher rate, suggesting that the original site was reacylated. Furthermore, tryptic digestion and peptide mapping showed that the same sites are acylated in vitro as in nerve slices indicating that the reaction has physiological significance. On incubation with [¹⁴C]palmitoyl-CoA, the synthetic peptide encompassing the natural P₀ acylation site (I¹⁵⁰RYCWLRR¹⁵⁷) was also spontaneously acylated at the cysteine residue. Thus, the integrity of the protein is not required for the nonenzymatic transacylation reaction. At pH 7.4 and 37°C, peptide palmitoylation followed a second-order reaction (k₂ = 246 ± 6 M^?1 min^?1) and is likely a bimolecular nucleophilic substitution with the peptide thiolate attacking the highly reactive thioester bond in palmitoyl-CoA. The activation energy calculated from the Arrhenius plot is ～2 kcal/mol and much lower than that of enzyme-catalyzed transacylations. Finally, two other P₀ peptides (V¹²¹PTRYG¹²⁶ and K¹⁰⁹TSQVTL¹¹⁵) as well as various unrelated thiol-containing compounds, including cysteine, glutathione, pressinoic acid (CYFQNC), and crustacean cardioactive peptide (PFCNAFTGC), were not autoacylated. These results indicate that the IRYCWLRR peptide represents a particular structural motif and/or has some chemical features that allow the reaction to occur spontaneously.     

S100A6 Competes with the TAZ2 Domain of p300 for Binding to p53 and Attenuates p53 Acetylation

S100A6 is a calcium binding protein that, like some other members of the S100 protein family, is able to bind p53. This interaction may be physiologically relevant considering the numerous connotations of S100 proteins and of S100A6, in particular, with cancer and metastasis. In this work, we show that the interaction with S100A6 is limited to unmodified or phosphorylated p53 and is inhibited by p53 acetylation. Using in vitro acetylation assay, we show that the presence of S100A6 attenuates p53 acetylation by p300. Furthermore, using ELISA, we show that S100A6 and the TAZ2 domain of p300 bind p53 with similar affinities and that S100A6 effectively competes with TAZ2 for binding to p53. Our results add another element to the complicated scheme of p53 activation.     

Binding Site and Inhibitory Mechanism of the Mambalgin-2 Pain-relieving Peptide on Acid-sensing Ion Channel 1a

Acid-sensing ion channels (ASICs) are neuronal proton-gated cation channels associated with nociception, fear, depression, seizure, and neuronal degeneration, suggesting roles in pain and neurological and psychiatric disorders. We have recently discovered black mamba venom peptides called mambalgin-1 and mambalgin-2, which are new three-finger toxins that specifically inhibit with the same pharmacological profile ASIC channels to exert strong analgesic effects in vivo. We now combined bioinformatics and functional approaches to uncover the molecular mechanism of channel inhibition by the mambalgin-2 pain-relieving peptide. Mambalgin-2 binds mainly in a region of ASIC1a involving the upper part of the thumb domain (residues Asp-349 and Phe-350), the palm domain of an adjacent subunit, and the β-ball domain (residues Arg-190, Asp-258, and Gln-259). This region overlaps with the acidic pocket (pH sensor) of the channel. The peptide exerts both stimulatory and inhibitory effects on ASIC1a, and we propose a model where mambalgin-2 traps the channel in a closed conformation by precluding the conformational change of the palm and β-ball domains that follows proton activation. These data help to understand inhibition by mambalgins and provide clues for the development of new optimized blockers of ASIC channels.     

Interactions of Chromatin Context,Binding Site Sequence Content,and Sequence Evolution in Stress-Induced p53 Occupancy and Transactivation

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10⁻⁷) and correlated with stronger p53RE sequences (p<10⁻¹¹⁰) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.     

The Role of Changing Loop Conformations in Streptavidin Versions Engineered for High-affinity Binding of the Strep-tag II Peptide

The affinity system based on the artificial peptide ligand Strep-tag® II and engineered tetrameric streptavidin, known as Strep-Tactin®, offers attractive applications for the study of recombinant proteins, from detection and purification to functional immobilization. To further improve binding of the Strep-tag II to streptavidin we have subjected two protruding loops that shape its ligand pocket for the peptide – instead of D-biotin recognized by the natural protein – to iterative random mutagenesis. Sequence analyses of hits from functional screening assays revealed several unexpected structural motifs, such as a disulfide bridge at the base of one loop, replacement of the crucial residue Trp120 by Gly and a two-residue deletion in the second loop. The mutant m1-9 (dubbed Strep-Tactin XT) showed strongly enhanced affinity towards the Strep-tag II, which was further boosted in case of the bivalent Twin-Strep-tag®. Four representative streptavidin mutants were crystallized in complex with the Strep-tag II peptide and their X-ray structures were solved at high resolutions. In addition, the crystal structure of the complex between Strep-Tactin XT and the Twin-Strep-tag was elucidated, indicating a bivalent mode of binding and explaining the experimentally observed avidity effect. Our study illustrates the structural plasticity of streptavidin as a scaffold for ligand binding and reveals interaction modes that would have been difficult to predict. As result, Strep-Tactin XT offers a convenient reagent for the kinetically stable immobilization of recombinant proteins fused with the Twin-Strep-tag. The possibility of reversibly dissociating such complexes simply with D-biotin as a competing ligand enables functional studies in protein science as well as cell biology.     

Systematic Mutational Analysis of Peptide Inhibition of the p53-MDM2/MDMX Interactions

Inhibition of the interaction between the tumor suppressor protein p53 and its negative regulators MDM2 and MDMX is of great interest in cancer biology and drug design. We previously reported a potent duodecimal peptide inhibitor, termed PMI (TSFAEYWNLLSP), of the p53-MDM2 and -MDMX interactions. PMI competes with p53 for MDM2 and MDMX binding at an affinity roughly 2 orders of magnitude higher than that of ^17-28p53 (ETFSDLWKLLPE) of the same length; both peptides adopt nearly identical α-helical conformations in the complexes, where the three highlighted hydrophobic residues Phe, Trp, and Leu dominate PMI or ^17-28p53 binding to MDM2 and MDMX. To elucidate the molecular determinants for PMI activity and specificity, we performed a systematic Ala scanning mutational analysis of PMI and ^17-28p53. The binding affinities for MDM2 and MDMX of a total of 35 peptides including 10 truncation analogs were quantified, affording a complete dissection of energetic contributions of individual residues of PMI and ^17-28p53 to MDM2 and MDMX association. Importantly, the N8A mutation turned PMI into the most potent dual-specific antagonist of MDM2 and MDMX reported to date, registering respective K_d values of 490 pM and 2.4 nM. The co-crystal structure of N8A-PMI-^25-109MDM2 was determined at 1.95 Å, affirming that high-affinity peptide binding to MDM2/MDMX necessitates, in addition to optimized intermolecular interactions, enhanced helix stability or propensity contributed by non-contact residues. The powerful empirical binding data and crystal structures present a unique opportunity for computational studies of peptide inhibition of the p53-MDM2/MDMX interactions.     

Both the Extracellular Leucine-Rich Repeat Domain and the Kinase Activity of FLS2 Are Required for Flagellin Binding and Signaling in Arabidopsis

In Arabidopsis, activation of defense responses by flagellin is triggered by the specific recognition of the most conserved domain of flagellin, represented by the peptide flg22, in a process involving the FLS2 gene, which encodes a leucine-rich repeat serine/threonine protein kinase. We show here that the two fls2 mutant alleles, fls2-24 and fls2-17, which were shown previously to confer insensitivity to flg22, also cause impaired flagellin binding. These features are rescued when a functional FLS2 gene is expressed as a transgene in each of the fls2 mutant plants, indicating that FLS2 is necessary for flagellin binding. The point mutation of the fls2-17 allele lies in the kinase domain. A kinase carrying this missense mutation lacked autophosphorylation activity when expressed in Escherichia coli. This indicates that kinase activity is required for binding and probably affects the stability of the flagellin receptor complex. We further show that overexpression of the kinase-associated protein phosphatase (KAPP) in Arabidopsis results in plants that are insensitive to flagellin treatment, and we show reduced flg22 binding in these plants. Furthermore, using the yeast two-hybrid system, we show physical interaction of KAPP with the kinase domain of FLS2. These results suggest that KAPP functions as a negative regulator of the FLS2 signal transduction pathway and that the phosphorylation of FLS2 is necessary for proper binding and signaling of the flagellin receptor complex.     

Small-Molecule Antagonists of p53-MDM2 Binding: Research Tools and Potential Therapeutics

p53 regulates a key pathway which protects normal tissues from tumor development that may result from diverse forms of stress. In the absence of stress, growth suppressive and proapoptotic activity of p53 is inhibited by MDM2 which binds p53 and negatively regulates its activity and stability. MDM2 antagonists could activate p53 and may offer a novel therapeutic approach to cancer. Recently, we identified the first potent and selective low molecular weight inhibitors of MDM2-p53 binding, the Nutlins. These molecules activate the p53 pathway and suppress tumor growth in vitro and in vivo. Nutlins represent valuable new tools for studying the p53 pathway and its defects in cancer. Their potent activity against osteosarcoma xenogrfts suggests that MDM2 antagonists may have a clinical utility in the treatment of tumors with wild-type p53.     

Modeling the therapeutic efficacy of p53 restoration in tumors

Although restoration of p53 function is an attractive tumor-specific therapeutic strategy, it remains unclear whether p53 loss is required only for transition through early bottlenecks in tumorigenesis or also for maintenance of established tumors. To explore the efficacy of p53 reinstatement as a tumor therapy, we used a reversibly switchable p53 knockin (KI) mouse model that permits modulation of p53 status from wild-type to knockout, at will. Using the well-characterized Emu-myc lymphoma model, we show that p53 is spontaneously activated when restored in established Emu-myc lymphomas in vivo, triggering rapid apoptosis and conferring a significant increase in survival. Nonetheless, reimposition of p53 function potently selects for emergence of p53-resistant tumors through inactivation of p19(ARF) or p53. Our study provides important insights into the nature and timing of p53-activating signals in established tumors and how resistance to p53 evolves, which will aid in the optimization of p53-based tumor therapies.     

Binding of p53 to the central domain of Mdm2 is regulated by phosphorylation

The Mdm2 protein is the major regulator of the tumor suppressor protein p53. We show that the p53 protein associates both with the N-terminal and with the central domain of Mdm2. The central p53-binding site of Mdm2 encompasses amino acids 235-300. Binding of p53 to the central domain is significantly enhanced after phosphorylation of the central domain of Mdm2. The N-terminal and central domains of Mdm2 act synergistically in binding to p53. p53 mutants that have mutations in the tetramerization domain and that fail to oligomerize do not show such an enhancement of binding in the presence of the other binding site.     

Both p53-PUMA/NOXA-Bax-mitochondrion and p53-p21cip1 pathways are involved in the CDglyTK-mediated tumor cell suppression

CDglyTK fusion suicide gene has been well characterized to effectively kill tumor cells. However, the exact mechanism and downstream target genes are not fully understood. In our study, we found that CDglyTK/prodrug treatment works more efficiently in p53 wild-type (HONE1) cells than in p53 mutant (CNE1) cells. We then used adenovirus-mediated gene delivery system to either knockdown or overexpress p53 and its target genes in these cells. Consistent results showed that both p53-PUMA/NOXA/Bcl2-Bax and p53-p21 pathways contribute to the CDglyTK induced tumor cell suppression. Our work for the first time addressed the role of p53 related genes in the CDglyTK/prodrug system.     

Micro-RNA Binding Site Polymorphisms in the WFS1 Gene Are Risk Factors of Diabetes Mellitus

The absolute or relative lack of insulin is the key factor in the pathogenesis of diabetes mellitus. Although the connection between loss of function mutations of the WFS1 gene and DIDMOAD-syndrome including diabetes mellitus underpins the significance of wolframin in the pathogenesis, exact role of WFS1 polymorphic variants in the development of type 1 and type 2 diabetes has not been discovered yet. In this analysis, 787 patients with diabetes and 900 healthy people participated. Genotyping of the 7 WFS1 SNPs was carried out by TaqMan assays. Association study was performed by χ²-test in combination with correction for multiple testing. For functional analysis, the entire 3’ UTR of the WFS1 gene was subcloned in a pMIR-Report plasmid and relative luciferase activities were determined. Linkage disequilibrium analysis showed a generally high LD within the investigated region, however the rs1046322 locus was not in LD with the other SNPs. The two miR-SNPs, rs1046322 and rs9457 showed significant association with T1DM and T2DM, respectively. Haplotype analysis also confirmed the association between the 3’ UTR loci and both disease types. In vitro experiments showed that miR-185 reduces the amount of the resulting protein, and rs9457 miRSNP significantly influences the rate of reduction in a luciferase reporter assay. Genetic variants of the WFS1 gene might contribute to the genetic risk of T1DM and T2DM. Furthermore demonstrating the effect of rs9457 in binding of miR-185, we suggest that the optimal level of wolframin protein, potentially influenced by miR-regulation, is crucial in normal beta cell function.     

Conformations of p53 response elements in solution deduced using site-directed spin labeling and Monte Carlo sampling

The tumor suppressor protein p53 regulates numerous signaling pathways by specifically recognizing diverse p53 response elements (REs). Understanding the mechanisms of p53-DNA interaction requires structural information on p53 REs. However, such information is limited as a 3D structure of any RE in the unbound form is not available yet. Here, site-directed spin labeling was used to probe the solution structures of REs involved in p53 regulation of the p21 and Bax genes. Multiple nanometer distances in the p21-RE and BAX-RE, measured using a nucleotide-independent nitroxide probe and double-electron-electron-resonance spectroscopy, were used to derive molecular models of unbound REs from pools of all-atom structures generated by Monte-Carlo simulations, thus enabling analyses to reveal sequence-dependent DNA shape features of unbound REs in solution. The data revealed distinct RE conformational changes on binding to the p53 core domain, and support the hypothesis that sequence-dependent properties encoded in REs are exploited by p53 to achieve the energetically most favorable mode of deformation, consequently enhancing binding specificity. This work reveals mechanisms of p53-DNA recognition, and establishes a new experimental/computational approach for studying DNA shape in solution that has far-reaching implications for studying protein–DNA interactions.     

Binding of Amphipathic Cell Penetrating Peptide p28 to Wild Type and Mutated p53 as studied by Raman,Atomic Force and Surface Plasmon Resonance spectroscopies

BackgroundMutations within the DNA binding domain (DBD) of the tumor suppressor p53 are found in > 50% of human cancers and may significantly modify p53 secondary structure impairing its function. p28, an amphipathic cell-penetrating peptide, binds to the DBD through hydrophobic interaction and induces a posttranslational increase in wildtype and mutant p53 restoring functionality. We use mutation analyses to explore which elements of secondary structure may be critical to p28 binding.MethodsMolecular modeling, Raman spectroscopy, Atomic Force Spectroscopy (AFS) and Surface Plasmon Resonance (SPR) were used to identify which secondary structure of site-directed and naturally occurring mutant DBDs are potentially altered by discrete changes in hydrophobicity and the molecular interaction with p28.ResultsWe show that specific point mutations that alter hydrophobicity within non-mutable and mutable regions of the p53 DBD alter specific secondary structures. The affinity of p28 was positively correlated with the β-sheet content of a mutant DBD, and reduced by an increase in unstructured or random coil that resulted from a loss in hydrophobicity and redistribution of surface charge.ConclusionsThese results help refine our knowledge of how mutations within p53-DBD alter secondary structure and provide insight on how potential structural alterations in p28 or similar molecules improve their ability to restore p53 function.General significanceRaman spectroscopy, AFS, SPR and computational modeling are useful approaches to characterize how mutations within the p53DBD potentially affect secondary structure and identify those structural elements prone to influence the binding affinity of agents designed to increase the functionality of p53.