透過結構分析探討大腸桿菌反應調節因子OmpR和其同源DNA的交互作用

Structural studies of response regulator OmpR and its interaction with the cognate DNA from Escherichia coli

蘇玄德(Sushant Sadotra)

指導教授 ： 陳金榜 ； 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(2021年)

摘要

EnvZ / OmpR兩成分系統控制了兩種膜孔蛋白OmpF和OmpC的表達，它們調節大腸桿菌內的摩爾滲透壓濃度。為了引起這種適應反應，EnvZ在信號轉導過程中將OmpR在N末端受體域（OmpRn）中保守的天門冬醯胺酸磷酸化。磷酸化的OmpRn會誘導OmpR C端去氧核糖核酸結合域（OmpRc）的構型改變，進而刺激去氧核糖核酸啟動子的識別，結合和活化轉錄。此外，OmpR還可以調節大腸桿菌或其他病原體中許多的持家基因和致病力基因，因此對於細菌的發病機制影響重大。然而對於 OmpR的構型或OmpRc-去氧核糖核酸的結合機制目前仍不清楚。在本研究中，我們解析了OmpRc與OmpF去氧核糖核酸啟動子F1區域的複合體晶體結構。並發現OmpRc獨特的結構域交換結構，在不同的結晶條件下均可觀察到。結構分析顯示，只有以傳統單體形式頭尾方向結合的OmpRc同源二聚體能結合其同源去氧核糖核酸，而結構域交換構型的OmpRc二聚體形式不能結合其同源去氧核糖核酸。我們還發現在結晶結構中，OmpR與特定序列的去氧核糖核酸鹼基有特異性接觸，這顯示OmpR可以作用在多個啟動子上。透過核磁共振光譜儀，螢光偏振和熱穩定性實驗等實驗結果，我們發現未磷酸化的全長OmpR也可以與其去氧核糖核酸啟動子結合，但與磷酸化的全長OmpR或單獨的OmpRc相比，結合親和力較弱。此發現證實磷酸化可以僅增強去氧核糖核酸的結合但不是必需的。此外，我們觀察到 OmpRc會以不對稱方式形成弱二聚體，這顯示未磷酸化OmpRc的去氧核糖核酸結合結構域之間可能具有弱相互作用，因此全長的OmpR可能需要磷酸化或與去氧核糖核酸結合才能正確的形成二聚體。整體而言，本研究展示了全長OmpR在水溶液中的性質, OmpRc獨特的結構域交換二聚狀態和OmpRc-去氧核糖核酸複合物中的二聚體界面。這些知識不僅讓我們對於OmpR的調節作用有更一步的理解，也能用於找尋可能的製藥標地。

關鍵字

EnvZ/OmpR兩成分系統 ； 蛋白質DNA 複晶體結構

並列摘要

EnvZ/OmpR two-component system controls the expression of two membrane porin proteins OmpF and OmpC that regulate the osmolarity levels in Escherichia coli. To elicit this adaptive response, EnvZ phosphorylates OmpR (OmpR-FL) at its conserved Asp residue in the N-terminal receiver domain (OmpRn) during signal transduction. Phosphorylation of OmpRn induces conformational changes in OmpR C-terminal DNA-binding domain (OmpRc) that further stimulates promoter DNA recognition, binding and transcription activation. Apart from that, OmpR is known to regulate many other housekeeping and virulence genes in E. coli and other pathogens and thus is highly responsible for bacterial pathogenesis. In spite of such biological importance, structural information of the OmpRc−DNA binding mechanism is still elusive. In our study, the crystal structure of OmpRc bound to the F1 region of ompF promoter DNA was determined. We also found that OmpRc in the apo-form has a unique domain-swapped structure that was observed in different crystallization conditions. Structural investigation showed that OmpRc could bind to the DNA only in the head-to-tail orientation of a homodimer formed by its traditional monomeric form but not by domain-swapped dimeric form. Furthermore, in the crystal structure, it was seen that DNA sequence-specific contacts of OmpRc with DNA bases were few upon DNA binding, suggesting that OmpR can be functional on multiple promoters. Using several biophysical examinations, such as NMR, thermal stability experiments, and fluorescent polarization we observed that unphosphorylated OmpR-FL could also bind to its promoter DNA, but with a weaker binding affinity compared to the phosphorylated OmpR-FL and OmpRc alone. This finding approves that phosphorylation may not be required but enhance DNA binding. Besides, we observed that OmpRc forms a weak dimer asymmetrically, suggesting that the DNA-binding domain of unphosphorylated OmpR may have weak interactions and therefore OmpR-FL may require phosphorylation or DNA binding for proper dimer formation.
Taken together, the solution behaviour of OmpR-FL, the unique domain-swapped dimeric state of OmpRc and the dimer interfaces in the OmpRc−DNA complex found in this study not only provide a better understanding of the regulatory role of OmpR but also identify the potential for this "druggable" target.

並列關鍵字

EnvZ/OmpR two-component system ； Protein-DNA complex crystal structure

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