The Relations Between Conformational Fluctuations In Transcription Factors And Transcriptional Activities: A Molecular Dynamics Based Study

In recent years, along with the development of calculator technique and software, the development of molecular mechanics, molecular dynamics, and quantum chemistry theories, via molecular modeling can build the three-dimensional structure of the macromolecules, to research the structure characteristics of the macromolecules, analyze the interaction between the receptor and ligand, describe protein biochemistry functions, and perform drug design and sieving, thus molecular simulation has already become a main research means in the fields of biology and medical science.Transcription factors are very necessary for RNA polymerase during transcription process. Studying recognition mechanism of transcription factor-DNA and the relationship between protein or protein-DNA conformational fluctuations (flexibility) and transcriptional activity is very important for clarifying the function and action mechanisms of transcription factor and useful for drug design. Our studying three transcription factors: homeodomain protein, ethylene-responsive element binding protein (EREBP) and heme activator protein has been well researched in bio-chemistry experimental works, but some pivotal problems involving in transcriptional activities still are unclear: homeodomain protein controls cell differentiation during the early stages of embryo development of many eukaryotic organisms. Both in vivo and in vitro studies show that the homeodomain protein is capable of sequence-specific binding. The residue 50 is a sequence discriminating residue, but the role of residue 50 in DNA recognition is still an unsettled issue; the ethylene-responsive element binding protein is very important for regulating plants to adapt exquisite environmental changes. During ethylene-responsive element binding protein binding DNA, the induced conformational changing relations between protein and DNA are also indistinct. These are very significant for understanding ethylene-responsive element binding protein transcriptional mechanism and plant pathology research; The function of heme activator protein (HAP) as a transcription factor is to regulate genes involved with oxidative phosphorylation and repairing, and regulation is mediated by the binding of HAP to the upstream activation sequences (UAS) of these target genes. The DNA-binding and transcriptional activation properties of the HAP1 protein are very interesting among the proteins containing a Zn2Cys6 domain. Of most interest in this regard are mutations at serine63 just N-terminal to the first cystenie of the Zn2Cys6 domain of HAP1. Two of the most unusual mutant forms are exhibited by the HAP1-18 (Ser→Arg substitution) and HAP1-PC7 (Ser→Gly substitution) proteins. While both proteins bind UAS_CYC7 site with near wild-type affinities, HAP1-18 shows elevated levels of transcriptional activation (10-100-fold), while HAP1-PC7 is transcriptionally silent. Some researches have reported the structure of the DNA-binding domain of wild-type HAP1, mutant HAP-18 and mutant HAP-PC7 bound to UASCYC7 and pointed out that differential transcriptional activities are due to alternative protein-DNA interactions.Although the application of these experimental methods is continually growing, they remain time-consuming and show limited applicability due to the difficulties of obtaining accurate experimental data. Molecular simulation can not only overcome the limiting of experimental tools effectively, but also guide the experiment. The application of molecular simulation to studying the interactions between ligand molecules and their protein receptors can afford theoretical guide for drug design or the structure modification of protein. In this thesis, we used molecular simulation technology to study above three transcriptional proteins that have significant medical value. The main results of the thesis are as follows:[1] Employing the crystal structure of free engrailed homeodomain and homeodomain-DNA complex as a starting structure we carried out MD simulations of: the complex between engrailed homeodomain and a 20 base-pair DNA containing TAATTA core sequence. The simulations show that homeodomain flexibility does not depend on its DNA ligation state. The engrailed homeodomain shows similar flexibility, and the recognition helix-3 shows very similar characteristic of high rigidity and limited conformational space in two complexation states (free engrailed homeodomain and homeodomain-DNA complex). At the same time, DNA structure has also no obvious conformational fluctuations. These results preclude the possibility of the side chain of Gln50 forming direct hydrogen bonds to the core DNA bases. MD simulation confirms a few well-conserved sites for water-mediated hydrogen bonds from protein to DNA are occupied by water molecules, and Gln50 interacts with corresponding core DNA bases through water-mediated hydrogen bonds.[2] The ethylene-responsive element binding protein as a novel fold for DNA recognition has been analyzed by means of molecular dynamics. The simulations show that the complex of protein-DNA trajectories show similar fluctuations in the atomic positions as uncomplexed, particularly at threeβstrands involving DNA binding. The calculations of entropy also affirm that GBD flexibility is basically similar for two ligation states. Further, the two complexation states(free engrailed ethylene-responsive element binding protein and ethylene-responsive element binding protein-DNA complex) present similar patterns of internal motions, indicating that the bound DNA can not alter GBD flexibility. It is inferred that the flexibility of GBD molecule is independent of its ligation state, while DNA shows better flexibility. So in the protein-DNA recognition, the GBD can induce DNA conformation changing to accomplish intermolecular recognition.[3] In order to further explore the unusual DNA-binding and transcriptional activation properties of the HAP1 protein, we now report the results of molecular dynamics (MD) simulations for UASCYC7 complexes with wild-type HAP1-wt, HAP1-18 and HAP1-PC7 and single DNA structure of UASCYC7, respectively. The results show that the protein-DNA interactions of three complexes accord with properties of their transcriptional activities as the experimental observations. The mutants at the position 63 in three HAPs can not change DNA binding modes due to similar internal motions, moreover, three bound DNA structures exhibit similar low flexibility, but three HAPs have different flexibility, particularly in N-term Arm and Zn2Cys6 Binuclear Cluster, which participate in DNA recognition. It is concluded that the difference of flexibilities in three HAPs results in diversities in conformations of N-term Arm and Zn2Cys6 Binuclear Cluster involving DNA recognition, causing varieties of protein-DNA interactions. According to these results, the flexibility of N-term and Zn2Cys6 Binuclear Cluster in HAP can play a crucial role in regulating transcriptional activation, which can directly lead to alternative protein-DNA interactions.