The Tumor Suppressor Protein P53dna Combined With The Research Domain And The Target Gene Interactions

The tumor suppressor protein p53 is a widely distributed phosphoprotein which functions to maintain the integrity of the genome. Wild-type p53 consists of three major functional domains, the C-terminal tetramerization domain, the N-terminal transactivation domain and the central DNA binding domain (p53DBD) encompassing amino acid residues from 96 to 308. It acts as a sequence-specific transcription factor to transactivate the downstream target genes which is activated in response to a variety of cell stress, and involves in many biological processes, such as cell cycle arrest, DNA repair, inhibition of angiogenesis, inhibition of metastasis and apoptosis. All of these presently known biological functions of p53 depend critically upon its DNA binding properties. Wild type p53 binds DNA through a sequence-specific DNA binding domain (p53DBD). p53 is mutated in more than half of the human cancers. The overwhelming majority of these mutations occurs in the sequence-specific DNA binding domain and result in the loss of its DNA binding activity. Therefore, site-specific recognition and DNA-binding activity of p53 are crucial for its tumor suppressor function.The crystal structure of p53DBD reveals that the p53 core domain structure consists of a beta sandwich that serves as a scaffold for two large loops and a loop-sheet-helix motif. Zn²⁺ is coordinated to three Cys (C176, C238 and C242) and a His (H179) in p53DBD. Zinc coordination is thought to be necessary for transcriptional activation and removal of zinc reduces the DNA-binding specificity. Nevertheless, the binding of mercury, cadmium and copper to the protein results in disrupting the p53 conformation and the DNA-binding activity. The opposite effects of metal ions on p53 support the notion that additional metal ions or cellular factors can affect specific recognition.In this paper, we investigated the binding reaction between p53DBD and nine kinds of metal ions by fluorescence titration method, the binding affinity of metal ions to p53DBD is Fe³⁺>Zn²⁺>Cu²⁺>Ca²⁺>Mg²⁺>Ba²⁺>Mn²⁺>Ni²⁺>Co²⁺. Analysis of the far-UV CD data clearly suggested that the binding of Ba²⁺, Ca²⁺, Co²⁺, Mn and Ni did not induce changes in protein secondary structure. The binding of Zn²⁺, Mg²⁺ and Fe³⁺ induced a subtle conformational change, while the binding of Cu²⁺ resulted in a lot of loss in its helical content. Analysis of ANS binding data showed that the binding of Mg²⁺ enhanced hydrophobic exposure on protein surface like Zn²⁺, while Fe³⁺ decreased the hydrophobic exposure. Therefore, Mg²⁺ and Fe³⁺ may be one of potential factors to affect or regulate the transactivation of p53.Then, we investigated the influence of Mg²⁺ on the Binding of p53DBD to DNA. Analysis of competitive binding revealed that magnesium competed with zinc for binding to p53DBD. Analysis of the CD data clearly suggested that the binding of magnesium ion induced a subtle conformational change rather than a radical modification of the overall protein architecture. Based on the results of electrophoretic mobility shift assays and fluorescence experiments, we concluded that the binding of Mg²⁺ stimulated the binding of the protein to DNA in a sequence-independent manner, which differed from that of zinc ions in a sequence-specific manner. Based on the facts that Mg²⁺ exists at relatively high concentration in the cell and several cellular enzymes can be regulated by Mg²⁺, we propose that Mg²⁺ is one of potential factors to affect or regulate the transactivation of p53.Studies on the effect of metal ions on the structural stability of p53DBD showed that the binding of metal ions increased the structural and thermal stability. Analysis of acrylamide quenching experiments revealed that the binding of metal ions to p53DBD induced a structural modification of the protein and this change provided significant protection against acrylamide quenching. Overall, we propose that metal ions play a dual modulatory role in the process of p53DBD functioning as a transcription factor. The metal ions not only support the DNA-binding affinity of p53DBD, but also stabilize the structure of the protein.Studies on the interaction between p53DBD and DNA by FRET showed that the bend angle of a half-site of p21 promoter induced by the binding of p53DBD was 23.6°.Studies on the interaction between p53DBD and the target genes in this paper provide us more precise information to understand the interaction between p53 and DNA on the single molecule lever. It is helpful to understand the mechanism of carcinogenesis of tumor cells and provide a help to the study and design of medicine.