克雷伯氏肺炎菌RstA反應調節蛋白質DNA結合區與雙股DNA複合體的結晶學結構研究

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研究生：	李易撰 Li, Yi-Chuan
論文名稱：	克雷伯氏肺炎菌RstA反應調節蛋白質DNA結合區與雙股DNA複合體的結晶學結構研究 Crystallographic study of the Klebsiella pneumoniae RstA DNA binding domain complex with double-stranded DNA
指導教授：	蕭傳鐙 Hsiao, Chwan-Deng 孫玉珠 Sun, Yuh-Ju
口試委員:	蕭傳鐙 Hsiao, Chwan-Deng 孫玉珠 Sun, Yuh-Ju 黃太煌 Huang, Tai-huang 陳金榜 Chen, Chinpan 殷献生 Yin, Hsien-Sheng
學位類別：	博士 Doctor
系所名稱：	生命科學暨醫學院 - 生物資訊與結構生物研究所 Institute of Bioinformatics and Structural Biology
論文出版年：	2014
畢業學年度：	102
語文別：	英文
論文頁數：	68
中文關鍵詞：	雙蛋白質調節系統、克雷伯氏肺炎菌
外文關鍵詞：	RstA, PmrA
相關次數：	點閱：2 下載：0
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當環境的化學分子或離子成分發生變化時，細菌通常藉由雙蛋白質調節系統(two-component system)以調控細胞內蛋白質表現，進而對環境變化做出反應。RstA/B則是對於環境中酸鹼值變化的調控蛋白質。酸性環境會讓細胞膜上的訊息受器RstB將細胞內的RstA磷酸化，活化後的RstA與特定DNA序列(RstA box)結合並開啟下游蛋白質表現，幫助形成細菌生物膜以在酸性環境中生存。RstA包含N端的磷酸接受區域(receiver domain, RD)與C端的DNA結合區域(DNA-binding domain,DBD)。磷酸接受區域活化後的RstA會以二聚體形式存在。我們以x-ray對晶體繞射分析解出克雷伯氏肺炎菌(Klebsiella pneumonia) RstA DBD和RstA box DNA 複合物的原子層次結構。另外，等溫滴定微量熱儀(isothermal titration calorimetry)的分析中顯示RstA與DNA結合模式為連續的，第一個RstA與DNA結合後會改變第二個RstA與DNA結合的強度。從結構與結合模式分析中，我們可以瞭解RstA二聚體提供一個平台讓第一個RstA DBD與特定DNA序列結合後，幫助第二個RstA結合並辨識正確的DNA序列。
PmrA/B是雙蛋白質調節系統中對於環境中含有抗生素多黏菌素(polymyxin)、鐵離子、鋁離子或微酸環境時的調節蛋白質。PmrA調控許多與改變細菌細胞壁上的脂多醣(lipopolysaccharides, LPS)組成有關的蛋白質。脂多醣組成改變後能有效地抵抗多黏菌素與宿主細胞產生的抗微生物胜肽。PmrA也是由N端的磷酸接受區域(receiver domain, RD)與C端的DNA結合區域(DNA-binding domain,DBD)所組成。磷酸接受區域活化後的PmrA會以二聚體形式存在。我們以x-ray對晶體繞射分析解出克雷伯氏肺炎菌PmrA和PmrA box DNA 複合物的原子層次結構。在此完整的PmrA和PmrA box DNA 複合物結構中，我們發現一新穎的磷酸接受區域對DNA結合區域的結合面，並且發現PmrA二聚體與DNA結合時是不對稱的。PmrA二聚體與DNA結合的不對稱性，也許與招攬RNA聚合酶時兩個PmrA單體會扮演不同角色有關。

The RstA/RstB system is a bacterial two-component regulatory system consisting of the membrane sensor, RstB, and its cognate response regulator (RR) RstA. The RstA of Klebsiella pneumoniae (kpRstA) consists of an N-terminal receiver domain (RD, residues 1-119) and a C-terminal DNA-binding domain (DBD, residues 130-236). Phosphorylation of kpRstA induces dimerization, which allows two kpRstA DBDs to bind to a tandem repeat, called the RstA box, and regulate the expression of downstream genes. Here we report the crystal structure of the kpRstA DBD/RstA box DNA complex. The structure of the kpRstA DBD/RstA box complex suggests that the upstream and downstream RstA DBDs interact with the RstA box in a different way. Combine with the equilibrium binding studies supported by Dr. Tai-huang Huang’s lab. The ITC analysis data revealed the two protomers within the kpRstA dimer bind to the RstA box in a sequential manner. Taken together, our results suggest a binding model where dimerization of the kpRstA RDs provides the platform to allow the first kpRstA DBD protomer to anchor protein-DNA interaction, whereas the second protomer plays a key role in ensuring correct recognition of the RstA box.
The PmrA/PmrB two-component system is the major regulator involved in the gene expression for lipopolysaccharides (LPS) modification in bacteria. PmrA is activated when the environment Fe3+, Al3+ and mild acidic environments. It activates genes including pbgPE, cptA and ugd can encode enzymes to change the composition of LPS. After modification, LPS can resist to polymyxin B and other host-derived antimicrobial peptides. The PmrA of Klebsiella pneumoniae (kpPmrA) is also consists of an N-terminal receiver domain (RD, residues 1-120) and a C-terminal DNA-binding domain (DBD, residues 126-226). Here we report the crystal structure of the kpPmrA/PmrA box DNA complex. Our intact response regulator complexed to DNA is asymmetric and reveals a novel heterodomain interface. A unique set of interactions between RD-DBD was observed. The asymmetric orientation of the PmrA dimer may play an important role in RNA polymerase recruitment through the upstream transactivation loop.

摘要..................................................I
Abstract................................................III
誌謝......................................................V
Content..................................................VI

Crystallographic study of the Klebsiella pneumoniae RstA DNA binding domain complex with double-stranded DNA

Chapter 1 Introduction......................................1
Chapter 2 Materials and Methods.............................5
2.1 Cloning, Protein Expression and Purification........................5
2.2 Crystallization and Data Collection...............................6
2.3 Structural Determination and Refinement...........................7
Chapter 3 Result...........................................9
3.1 Overall Crystal Structures of RstA DBD/DNA complex.................9
3.2 Upstream and downstream RstA DBD proteins interactions...............9
3.3 kpRstA DBD-dsDNA interactions...............................11
3.4 Comparison of RstA and PhoB complexes.........................13
3.5 Structural Changes upon DNA Binding...........................14
Chapter 4 Discussion.......................................16
Tables...................................................21
Table 1. X-ray Diffraction Data and Structure Refinement Statistics...........21
Figures..................................................22
Figure 1. Model of the PhoPQ-RstAB signal relay cascade for transcription regulation of the stress response genes......................................22
Figure 2. Sequence comparison of kpRstA with other response regulators from the OmpR/PhoB subfamily.........................................23
Figure 3. SDS PAGE analysis of RstA and RstA DBD/DNA complex crystal....................................................24
Figure 4. Electron density map of RstA-DBD/DNA-23 complex.............25
Figure 5. X-ray crystal structure of RstA-DBD/DNA-23 complex ............26
Figure 6. Comparison of the RstA-DBA/DNA-23 complex to the PhoB-DNA complex...................................................27
Figure 7. The residues involved in the DBD-DBD interactions..............28
Figure 8. The residues involved in the upstream protomer-DNA interactions.....29
Figure 9. Sketch of the interactions between RstA DBD and RstA box DNA.....30
Figure 10. Conformational changes of RstA DBD when binding to RstA box....31
Figure 11. Determination of binding isotherm of kpRstA DBD with various RstA box DNA by isothermal titration calorimetry. (From Dr. Tai-huang Huang’s lab.) ....32
Figure 12. The model of response regulator RstA binding to the promoter RstA box......................................................33


Crystallographic study of the Klebsiella pneumoniae PmrA complex with double-stranded DNA

Chapter 1 Introduction.....................................34
Chapter 2 Materials and Methods............................37
2.1 Cloning, Protein Expression and Purification.......................37
2.2 Crystallization and Data Collection..............................37
2.3 Structural Determination and Refinement..........................38
Chapter 3 Result..........................................40
3.1 Overall Structures of PmrA-DNA complex.........................40
3.2 kpPmrA-dsDNinteractions....................................42
3.3 RD dimer interface and active site...............................43
3.4 Upstream and downstream PmrA DBD interactions...................44
Chapter 4 Discussion.......................................46
Tables...................................................48
Table 1. X-ray Diffraction Data and Structure Refinement Statistics...........48
Figures..................................................49
Figure 1. Model of the interactions between the PhoP/PhoQ and PmrA/PmrB systems in Klebsiella pneumoniae.......................................49
Figure 2. Structures of OmpR/PhoB family...........................50

Figure 3. Sequence comparison of kpPmrA with other response regulators from the OmpR/PhoB subfamily.........................................51
Figure 4. SDS PAGE analysis of PmrA and crystals of PmrA-26bp and PmrA-25bp.................................................52
Figure 5. Electron density map of PmrA/DNA-25bp complex...............53
Figure 6. X-ray crystal structure of PmrA-DBD/DNA-25 complex...........54
Figure 7. Sketch of the interactions between PmrA DBD and PmrA box DNA....55
Figure 8. Residues involved in PmrA DBD-DBD interactions...............56
Figure 9. Salt bridges between the RD-RD interface.....................57
Figure 10. Hydrophobic interactions between the RD-RD interface...........58
Figure 11. Active site interaction residues............................59
Figure 12. The residues involved in the RD-DBD interactions...............60
Figure 13. Residues orientation changed during the formation of RD-DBD interface...................................................61
Figure 14. The superimposed of KpPmrA and EcoKdpE..................62
Figure 15. The model of KpPmrA bind to sigma 70......................63
Figure 16. Proposed model for transcription activation by PmrA.............64
References...............................................65

                                

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