Over-expression of the human MDM2 p53 binding domain by fusion to a p53 transactivation peptide

MDM2 binds to the tumor suppressor protein p53 and regulates the level of p53 in cells. Although it is possible to prepare a small amount of the region of MDM2 that binds to p53, the expression level of this fragment of MDM2 is relatively low, limiting the studies involving this protein. Here, we describe a construct for the optimized bacterial expression and purification of the MDM2 p53 binding domain. We found that the expression level of the soluble MDM2 p53 binding domain in bacteria was increased dramatically by fusing it to its interaction partner, the p53 transactivation peptide. Attachment of the p53 transactivation peptide (residues 17-29) to the N-terminus of MDM2 resulted in a more than 200-fold increase of soluble protein expression of the p53 binding domain in bacteria. To obtain the final MDM2 p53 binding domain (residues 5-109) we inserted a tobacco etch virus protease recognition site between the P53 peptide and the MDM2 p53 binding domain. To weaken the protein/peptide interaction and facilitate the separation of the protein from the complex, we introduced a point mutation of one of the key interaction residues (F19A or W23A) in the p53 peptide. The advantages of our new construct are high yield and easy purification of the MDM2 protein.     

Effects of oncogenic mutations and DNA response elements on the binding of p53 to p53-binding protein 2 (53BP2)

The tumor suppressor p53 is frequently mutated in human cancers. Upon activation it can induce cell cycle arrest or apoptosis. ASPP2 can specifically stimulate the apoptotic function of p53 but not cell cycle arrest, but the mechanism of enhancing the activation of pro-apoptotic genes over cell cycle arrest genes remains unknown. In this study, we analyzed the binding of 53BP2 (p53-binding protein 2, the C-terminal domain of ASPP2) to p53 core domain and various mutants using biophysical techniques. We found that several p53 core domain mutations (R181E, G245S, R249S, R273H) have different effects on the binding of DNA response elements and 53BP2. Further, we investigated the existence of a ternary complex consisting of 53BP2, p53, and DNA response elements to gain insight into the specific pro-apoptotic activation of p53. We found that binding of 53BP2 and DNA to p53 is mutually exclusive in the case of GADD45, p21, Bax, and PIG3. Both pro-apoptotic and non-apoptotic response elements were competed off p53 by 53BP2 with no indication of a ternary complex.     

Thermodynamics of peptide inhibitor binding to HIV-1 gp41

The gp41 subunit of the human immunodeficiency virus type 1 envelope glycoprotein mediates fusion of the cellular and viral membranes. The gp41 ectodomain is a trimer of alpha-helical hairpins, where N-terminal helices form a parallel three-stranded coiled-coil core and C-terminal helices pack around the core. A deep hydrophobic pocket on the N-terminal core represents an attractive target for antiviral therapeutics. We have employed a soluble derivative of the gp41 core ectodomain and small cyclic disulfide D-peptide inhibitors to define the stoichiometry, affinity, and thermodynamics of ligand binding to this pocket using isothermal titration calorimetry. These inhibitors bind with micromolar affinity to the pocket with the expected stoichiometry of three peptides per gp41 core trimer. There are no cooperative interactions among the three binding sites. Linear eight- or nine-residue D-peptides derived from the pocket-binding domain of the cyclic molecules also bind specifically. A negative heat capacity change is observed and is consistent with burial of hydrophobic surface upon binding. Contrary to expectations for a reaction dominated by the classical hydrophobic effect, peptide binding is enthalpically driven and is opposed by an unfavorable negative entropy change. The calorimetry data support models whereby dominant negative inhibitors bind to a transiently exposed surface on the prefusion intermediate state of gp41 and disrupt subsequent resolution to the fusion-active six-stranded hairpin conformation.     

Allosteric effects mediate CHK2 phosphorylation of the p53 transactivation domain

The tumour suppressor p53 is a tetrameric protein that is phosphorylated in its BOX-I transactivation domain by checkpoint kinase 2 (CHK2) in response to DNA damage. CHK2 cannot phosphorylate small peptide fragments of p53 containing the BOX-I motif, indicating that undefined determinants in the p53 tetramer mediate CHK2 recognition. Two peptides derived from the DNA-binding domain of p53 bind to CHK2 and stimulate phosphorylation of full-length p53 at Thr 18 and Ser 20, thus identifying CHK2-docking sites. CHK2 can be fully activated in trans by the two p53 DNA-binding-domain peptides, and can phosphorylate BOX-I transactivation-domain fragments of p53 at Thr 18 and Ser 20. Although CHK2 has a basal Ser 20 kinase activity that is predominantly activated towards Thr 18, CHK1 has constitutive Thr 18 kinase activity that is predominantly activated in trans towards Ser 20. Cell division cycle 25C (CDC25C) phosphorylation by CHK2 is unaffected by the p53 DNA-binding-domain peptides. The CHK2-docking site in the BOX-V motif is the smallest of the two CHK2 binding sites, and mutating certain amino acids in the BOX-V peptide prevents CHK2 activation. A database search identified a p53 BOX-I-homology motif in p21^WAF1 and although CHK2 is inactive towards this protein, the p53 DNA-binding-domain peptides induce phosphorylation of p21^WAF1 at Ser 146. This provides evidence that CHK2 can be activated allosterically towards some substrates by a novel docking interaction, and identify a potential regulatory switch that may channel CHK2 into distinct signalling pathways in vivo.     

Thermodynamics of peptide binding to the transporter associated with antigen processing (TAP)

The ATP-binding cassette (ABC) transporter TAP plays an essential role in antigen processing and immune response to infected or malignant cells. TAP translocates proteasomal degradation products from the cytosol into the endoplasmic reticulum, where MHC class I molecules are loaded with these peptides. Kinetically stable peptide-MHC complexes are transported to the cell surface for inspection by cytotoxic T lymphocytes. The transport cycle of TAP is initiated by peptide binding, which is responsible for peptide selection and for stimulation of ATP-hydrolysis and subsequent translocation. Here we have analysed the driving forces for the formation of the peptide-TAP complex by kinetic and thermodynamic methods. First, the apparent peptide association and dissociation rates were determined at various temperatures. Strikingly, very high activation energies for apparent association (E(a)(ass)=106 kJmol(-1)) and dissociation (E(a)(diss)=80 kJmol(-1)) of the peptide-TAP complex were found. Next, the temperature-dependence of the peptide affinity constants was investigated by equilibrium-binding assays. Along with calculations of free enthalpy deltaG, enthalpy deltaH and entropy deltaS, a large positive change in heat capacity was resolved (deltaC degrees =23 kJmol(-1)K(-1)), indicating a fundamental structural reorganization of the TAP complex upon peptide binding. The inspection of the conformational entropy reveals that approximately one-fourth of all TAP residues is rearranged. These thermodynamic studies indicate that at physiological temperature, peptide binding is endothermic and driven by entropy.     

Akt-dependent phosphorylation of p27Kip1 promotes binding to 14-3-3 and cytoplasmic localization

In many human cancers, the cyclin-dependent kinase inhibitor p27(Kip1) is expressed at low or undetectable levels. The decreased p27(Kip1) expression allows cyclin-dependent kinase activity to cause cells to enter into S phase and correlates with poor patient survival. Inhibition of serine/threonine kinase Akt signaling by some pharmacological agents or by PTEN induces G(1) arrest, in part by up-regulating p27(Kip1). However, the role of Akt-dependent phosphorylation in p27(Kip1) regulation is not clear. Here, we show that Akt bound directly to and phosphorylated p27(Kip1). Screening p27(Kip1) phosphorylation sites identified the COOH-terminal Thr(198) residue as a novel site. Further analysis revealed that 14-3-3 proteins bound to p27(Kip1) through Thr(198) only when it was phosphorylated by Akt. Although Akt also phosphorylated p27(Kip1) at Ser(10) and Thr(187), these two sites were not involved in the binding to 14-3-3 proteins. p27(Kip1) phosphorylated at Thr(198) exists only in the cytoplasm. Therefore, Akt promotes cell-cycle progression through the mechanisms of phosphorylation-dependent 14-3-3 binding to p27(Kip1) and cytoplasmic localization.     

Apak competes with p53 for direct binding to intron 1 of p53AIP1 to regulate apoptosis

The KRAB-type zinc-finger protein Apak was recently identified as a negative regulator of p53-mediated apoptosis. However, the mechanism of this selective regulation is not fully understood. Here, we show that Apak recognizes the TCTTN₂₋₃₀TTGT consensus sequence through its zinc-fingers. This sequence is specifically found in intron 1 of the proapoptotic p53 target gene p53AIP1 and largely overlaps with the p53-binding sequence. Apak competes with p53 for binding to this site to inhibit p53AIP1 expression. Upon DNA damage, Apak dissociates from the DNA, which abolishes its inhibitory effect on p53-mediated apoptosis.     

Generation of monospecific peptide antibodies to the DNA binding domain of p53

The DNA binding domain (DBD) is the most mutated region of p53 in tumors and has proven to be relatively resistant to the generation of specific antibodies. Template assembled synthetic peptide (TASP) synthesis of a peptide derived from the DBD creates a highly immunogenic molecule without the need for large carriers such as keyhole limpet hemocyanin (KLH). In addition, a rapid means of generating monoclonal antibodies can be achieved through immunization in conjunction with ABL/MYC retrovirus injection into recipient mice. In this paper, we demonstrate that an antibody generated by this means, KH2, reacts specifically with the DBD of p53. To date, this is the first example of a peptide immunogen used successfully in ABL/MYC monoclonal antibody production. KH2 is also the first example of a monospecific antibody that directly binds to and, by definition, assumes the conformation of the DNA binding region of p53.     

Modulation of p53 binding to Holliday junctions and 3-cytosine bulges by phosphorylation events

Recognition of certain types of DNA lesions by the tumor suppressor protein, p53, represents one of the several downstream functions of this protein in response to DNA damage. This binding property is regulated by several factors including posttranslational modifications and interactions with other proteins. Phosphorylation by several stress-response kinases activates p53 by increasing protein stability as well as transactivation properties. Here we examined the effect of phosphorylation events on the sequence-independent binding properties of p53 using two DNA substrates: One resembling Holliday junctions and the other containing extra base bulges. Gel retardation assays showed that dephosphorylation of serine 392 in the C-terminal domain of p53 greatly reduces Holliday junction and lesion recognition. In contrast, sequence-specific binding is disrupted by the removal of some N-terminal phosphates but not serine 392. Rephosphorylation of p53 by certain kinases can restore p53 recognition of Holliday junctions and 3-cytosine bulges. In all cases, phosphorylation of serine 392 occurs; however, reactivation also involves other residues. Together, the results show that p53 DNA binding activity is strongly regulated by the phosphorylation state of the protein.     

Design of a novel MDM2 binding peptide based on the p53 family

p53 is a major tumor suppressor protein, that binds to, and is negatively regulated by MDM2. In tumors over expressing MDM2, p53 function can be rescued through the disruption of the MDM2-p53 interactions by small molecules and peptides. It is known that MDM2 also binds p73 but not p63, the two homologues of p53. We dissect the structural and energetic reasons underlying this discrimination and have identified a peptide that is intrinsically less helical than p53 and yet has a higher affinity for MDM2. The increased disorder has been introduced by localizing a cationic residue in between two anionic residues, imparting a degree of frustration to the system. In addition, the introduction of a bulkier hydrophobic group towards the centre of the peptide enables the peptide to adapt a bound conformation that on the one hand is most strained, and yet enables the peptide to straddle the largest surface of MDM2, amongst all the peptides. Computations also reveal that this peptide is a dual inhibitor, binding to MDMX. The computed affinity of the new peptide has been validated against MDM2 using fluorescence-based thermal shift assays.     

LZAP inhibits p38 MAPK (p38) phosphorylation and activity by facilitating p38 association with the wild-type p53 induced phosphatase 1 (WIP1)

LZAP (Cdk5rap3, C53) is a putative tumor suppressor that inhibits RelA, Chk1 and Chk2 and activates p53. LZAP is lost in a portion of human head and neck squamous cell carcinoma and experimental loss of LZAP expression is associated with enhanced invasion, xenograft tumor growth and angiogenesis. p38 MAPK can increase or decrease proliferation and cell death depending on cellular context. LZAP has no known enzymatic activity, implying that its biological functions are likely mediated by its protein-protein interactions. To gain further insight into LZAP activities, we searched for LZAP-associated proteins (LAPs). Here we show that the LZAP binds p38, alters p38 cellular localization, and inhibits basal and cytokine-stimulated p38 activity. Expression of LZAP inhibits p38 phosphorylation in a dose-dependent fashion while loss of LZAP enhances phosphorylation and activation with resultant phosphorylation of p38 downstream targets. Mechanistically, the ability of LZAP to alter p38 phosphorylation depended, at least partially, on the p38 phosphatase, Wip1. Expression of LZAP increased both LZAP and Wip1 binding to p38. Taken together, these data suggest that LZAP activity includes inhibition of p38 phosphorylation and activation.     

Coupling of the antennapedia third helix to a potent antagonist of the p53/hdm2 protein-protein interaction

The use of cell-membrane translocating sequences for intracellular delivery of peptides can be a powerful approach to validate drug discovery targets in cellular settings. To accomplish this, a protocol has been implemented to couple the antennapedia third helix (residues 43-58) to a potent antagonist of the p53/hdm2 protein-protein interaction without affecting its in vitro inhibitory activity.     

N-Acylpolyamine inhibitors of HDM2 and HDMX binding to p53

Selective inhibition of protein–protein interactions important for cellular processes could lead to the development of new therapies against disease. In the area of cancer, overexpression of the proteins human double minute 2 (HDM2) and its homolog HDMX has been linked to tumor aggressiveness. Both HDM2 and HDMX bind to p53 and prevent cell cycle arrest or apoptosis in damaged cells. Developing a strategy to simultaneously prevent the binding of both HDM2 and HDMX to p53 is an essential feature of inhibitors to restore p53 activity in a number of different cancers. Inhibition of protein–protein interactions with synthetic molecules is an emerging area of research that requires new inhibitors tailored to mimic the types of interfaces between proteins. Our strategy to create inhibitors of protein–protein interactions is to develop a non-natural scaffold that may be used as a starting point to identify important molecular components necessary for inhibition. In this study, we report an N-acylpolyamine (NAPA) scaffold that supports numerous sidechains in a compact atomic arrangement. NAPAs were constructed by a series of reductive aminations between amino acid derivatives followed by acylation at the resulting secondary amine. An optimized NAPA was able to equally inhibit the association of both HDM2 and HDMX with p53. Our results demonstrate some of the challenges associated with targeting multiple protein–protein interactions involved in overlapping cellular processes.