DNA binding and functional association network analyses of the transcriptional regulator protein WhiA (Rv1423) from Mycobacterium tuberculosis H37Rv = 결핵균 H37Rv 유래 전사 조절 단백질 WhiA(Rv1423)의 DNA 결합 및 기능적 연관 네트워크 분석

다국어 초록 (Multilingual Abstract)

DNA binding and functional association network analyses of the transcriptional regulator protein WhiA (Rv1423) from Mycobacterium tuberculosis H37Rv Jeong, Daeun Department of Clinical Laboratory Science Graduate School, Catholic University of Pusan Advisor : Professor Lee, Ki Seog, Ph.D. Mycobacterium tuberculosis persists within host cells under nutrient-limited conditions, suggesting that it possesses adaptive mechanisms to regulate growth and metabolism in response to environmental changes. M. tuberculosis Rv1423 (MtWhiA) is a transcriptional regulatory protein that may be involved in the regulations of cell membrane homeostasis and cell division. Although previous studies have focused on the role of WhiA in spore-forming bacteria, more detailed studies are required to understand the expanded functional role of MtWhiA as a non-spore-forming bacteria. This study aims to investigate the binding properties of MtWhiA to DNA through biochemical analysis using homology modeling, protein-DNA docking simulations and site-directed mutagenesis. The binding affinity of DNA containing the biding motif (GACACACC) to MtWhiA was estimated to be a dissociation constant (KD) of 55.0 nM, indicating that the binding signal for MtWhiA increased in a concentration-dependent manner of the fluorescence-labeled DNA. Moreover, the C-terminal domain of MtWhiA exhibited an approximately two-fold lower DNA-binding affinity compared to the full-length MtWhiA, implying that both domains may contribute cooperatively to DNA binding. In the macromolecular docking using homology modeling of MtWhiA and DNA, the best-fit model of MtWhiA-DNA complex was estimated a binding free energy of –5.8 kcal/mol and predicted that residues R177, R185, K287, and D288 may be involved in the binding of DNA motif. Mutational studies revealed that the binding efficiency of R177A, K287A, and D288A mutants was an approximately 2~3-fold lower than that of the wild-type. In particular, the R185A mutant was shown to significantly affect the DNA binding. Furthermore, functional protein-protein network analysis was suggested that MtWhiA may be associated with genes involved in cell envelope homeostasis and cell division, such as Rv1420-1422, whiB, and ftsZ. Taken together, these results provide biochemical insights into the interactions between the transcriptional regulator MtWhiA and DNA, including the identification of residues that interact with DNA motif. Key word(s) : Mycobacterium tuberculosis, Rv1423, Transcriptional regulator, DNA binding, Functional network analysis

목차 (Table of Contents)

• Ⅰ. Introduction 1
• Ⅱ. Materials and methods 3
• 1. Gene cloning and protein expression
• 2. Protein purification
• 3. DNA binding assay
• 4. Analysis of binding affinity
• 5. Homology modeling and protein-DNA docking simulation
• 6. Preparation of MtWhiA mutants
• 7. Functional Network Analysis
• Ⅲ. Results 13
• 1. Gene cloning and protein purification of MtWhiA and MtWhiA-CTD
• 2. Binding properties of MtWhiA-FL and–CTD with DNA motif oligomer
• 3. Homology modeling and secondary structure comparison of MtWhiA
• 4. Macromolecular docking and structural comparisons of MtWhiA in complex with DNA
• 5. Identification of key residues involved in the binding of DNA to MtWhiA
• 6. Functional relationship of MtWhiA with neighborhood proteins
• Ⅳ. Discussion 31
• References 33
• Abstract in Korean 36