Interfacial properties of p53-DNA complexes containing various recognition elements

Highlights

• DNA binding to oncoprotein p53 was detected by constant current chronopotentiometry.

• Surface-attached DNA-p53 complexes on mercury disintegrate at negative potentials.

• Complexes of p53 core domain with various sequence-specific DNA were discriminated.

Abstract

Methods which can distinguish between specific and non-specific protein interactions leading to the identification of hubs and nodes are still desired. This work shows utilization of chronopotentiometric stripping analysis in combination with a mercury electrode in the study of protein-DNA interactions at thiol-modified electrodes. The complex of tumor suppressor p53 core domain (p53CD) and DNA undergoes disintegration due to the effect of the electric field, accompanied by a remarkable increase in the electrocatalytic reduction signal. By adjusting stripping current intensities and temperature, the transition between intact and disintegrated complex reflected differences in the stabilities of sequence-specific complexes with different recognition elements. Higher stabilities of p53-DNA complexes were observed for DNA binding sites connected with cell-cycle arrest and p53 negative autoregulation, than those for DNA associated with cell apoptosis, in good concordance with electrophoretic mobility shift assay in polyacrylamide gels. These data highlight the utility of this method for studying the dynamics of surface-attached protein-DNA complexes.

Introduction

Protein–DNA interactions help to understand, which transcription factor binds to which promoter, resolve promoter structure, identify a transcription factor binding box, etc. Hence the truly importance is understanding how regulatory proteins interact with the genetic material in the proper way [1]. The list of challenges comes from our health problems associated with the recent extension of life expectancy, such as type II diabetes, cardiovascular problems, neurodegeneracy, cancer, etc. [2]. The relevance of DNA–protein interactions is not limited to biology, medicine, or pharmacology, but they are also widely used in biotechnology. Relatively simple, rapid, and extremely sensitive in vitro methods are still sought. New ways, allowing to study where, when, and to what extent proteins interact with DNA with an unprecedented precision, are required on a larger scale more than ever [3]. Electrochemistry as time- and cost-containing method can be applied for DNA-protein interaction studies [4]. Up to now, electrochemical works predominantly dealt with aptamers (reviewed in refs. [5, 6]) and mostly used labeled DNA or protein. Label-free electrochemical determination of protein-DNA complexes appears also possible because both DNA [6] and proteins [4,7] are electrochemically active.

Transcription factor p53 protein operates as a tumor suppressor and helps to regulate hundreds of genes in response to various types of stress, such as UV radiation, DNA damage, and hypoxia [8]. The tumor suppressor protein p53, once activated depending on the cellular environment [8,9], results in cell-cycle arrest, DNA repair, senescence and apoptosis [10,11] through transactivation of target genes containing p53 DNA binding sites. DNA binding is critical for the biological functions of p53 [12]. The consensus sequence for p53 binding contains two copies of the half-site 10-base pair motif RRRC(A/T)|(T/A)GYYY separated by up to 13 bp (R represents purines while Y stands for pyrimidines in this sequence; the center of symmetry within the half site is indicated by the vertical bar) [13]. Four molecules of p53 bind to the full-length recognition element in double-stranded DNA (dsDNA) stabilized by both intradimer and interdimer interactions mediated by the p53 core domains (p53CD) [14]. The sequence and structural features of the p53 binding sites provide for p53 recruitment to target sites [15]. The binding of activated tumor suppressor protein to p53 response element leads to diverse cellular outcomes, such as the well-known MDM2 regulation, DNA repair, cell-cycle arrest and/or apoptosis [10,11]. How p53 chooses which genes to activate is critical in understanding its role as a tumor suppressor. Several studies showed that most of the high affinity DNA sequences are from regulatory regions of p53 target genes involved in cell-cycle arrest, DNA repair, and p53 negative autoregulation [16,17], while sequences from genes associated with apoptosis show low affinity [14]. The intrinsic structural properties of the DNA sequence play a role in modulating the affinity of p53 for its binding sites [14,16,17].

The 393-amino acid p53 protein has a complex domain structure, which comprises well-defined domains and natively unfolded regions [18]. Various p53-DNA complexes have been studied by different methods including fluorescence anisotropy, NMR, MS, electrophoretic mobility shift assay (EMSA) etc. [[18], [19], [20]]. Electrochemical methods utilizing the intrinsic signals of proteins [4] or DNA [21,22] are rarely used for the study of protein-nucleic acid complexes in general [4,6], in spite of the advantages of these methods, such as simplicity, rapidity, easy miniaturization, low cost and the small amounts of protein required. The electrochemical behavior of p53 core domain (p53CD, 94-312 aminno acids), responsible for specific interaction of p53 with dsDNA, was described using chronopotentiometric stripping (CPS) analysis on a dithiothreitol (DTT)-modified hanging mercury drop electrode (HMDE) [23] by so-called peak H, which is sensitive to structural changes in proteins [4,[23], [24], [25]]. CPS data for p53CD wild type and mutant forms exhibited good correlation with mass spectroscopy (MS) and nuclear magnetic resonance (NMR) structural data [18,26,27] and provided information about protein structural dynamics at the surface. The CPS analysis appeared suitable not only for the study of free p53CD protein, but also to study p53CD complex with DNA containing the consensus sequence, DNA_CON [28]. We have demonstrated that the CPS analysis performed with a thiol-modified mercury electrode [24,25] can be used to distinguish between specific and nonspecific binding of p53 protein to dsDNA [28] by following the dependences of CPS peak H height on stripping current intensity (I_str) and temperature. In protein-DNA complexes, the structural transitions of the specific protein-DNA binding occurred at different I_str and temperature values from those observed with nonspecific protein-DNA binding. Similar electrochemical behavior was found for the complex between a smaller monomeric hydrolase-lysozyme and single stranded DNA aptamer [29] at conditions under which thioglycolic acid (TGA) was used instead of DTT. TGA as an alkanethiol bearing a negatively charged terminal group could eliminate adsorption of ssDNA to the electrode surface better than DTT.

In our previous paper [28] using CPS we distinguished the p53 sequence specific DNA binding from binding to non-specific sequence at a DTT-modified HMDE. To prevent direct contact of p53CD and DNA with the mercury surface, we used alkanethiol dithiothreitol (DTT) creating a self-assembled monolayer at HMDE surface. We chose DTT because this reducing agent is frequently added to protein solutions at millimolar concentrations to keep proteins, in our case p53CD, in its native reduced state. Here we show that our CPS method is suitable for the solving of biological problems, such as for discerning p53CD-DNA complexes containing different DNA consensus sequences (DNA_CON) and their stabilities. In addition, we used Electrochemical Impedance Spectroscopy (EIS) to learn more about the properties of the p53CD-DNA complex at the charged interface. Using EIS it was not possible to discriminate between specific and nonspecific p53CD-DNAs, neither at a bare nor DTT-modified HMDE. Nevertheless the combination of EIS with CPS helped us to better understand the behavior of the free p53CD protein and its complexes on the charged interfaces. The method utilizing CPSis based on the disintegration of the p53CD-DNA complex due to the exposure of the surface-attached complex to negative potentials. Using appropriate current density, this disintegration can be prevented by minimizing the time for which the surface-attached p53CD-DNA complex is exposed to negative potentials. Here we show that the stabilities of p53CD forming complexes with DNA binding sites involved in cell-cycle arrest (DNA_p21), p53 negative autoregulation (DNA_MDM2) and apoptosis (DNA_puma, DNA_bax) can be distinguished by CPS. Our CPS results were in good correlation with the data obtained by electrophoretic mobility shift assay.