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

李易撰(Yi-Chuan Li)

指導教授 ： 蕭傳鐙 ； 孫玉珠

國立清華大學/生命科學院/生物資訊與結構生物研究所/博士(2014年)

摘要

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。

關鍵字

雙蛋白質調節系統 ； 克雷伯氏肺炎菌

並列摘要

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.

並列關鍵字

RstA ； PmrA ； two-component system ； Klebsiella pneumoniae

參考文獻

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