克雷白氏肺炎桿菌 (Klebsiella pneumoniae) 為院內感染常見的病原菌之一，在此菌的細胞外膜上具有大量且複雜的莢膜多醣體 (capsular polysaccharide, CPS)，用以躲避宿主的吞噬作用(Phagocytosis) 以提高存活率及感染力。在此細菌中，調控CPS生合成之基因 (cps genes)由雙分子系統感應蛋白RcsC與調節蛋白 RcsB所參與，RcsB在此雙分子系統中的必要性已被確立但其調控機制仍未被清楚的探討。本篇研究旨在探討磷酸化對於RcsB調控cps genes之影響，藉由模擬磷酸化之突變型D56E及野生型WT結構與功能之比較，建構RcsB被磷酸化後的活化態對cps genes之調控機制。利用電泳凝膠位移測定 (EMSA)，我們發現D56E對於 cps genes啟動子具有較高的結合能力，意味著磷酸化後的RcsB能夠顯著地增加cps genes的表現量。此外，從四級結構的分析中發現，D56E具有四聚體 (tetramer)的存在，此四聚體不僅能夠增加RcsB對於 cps genes啟動子的結合能力，更能有效地提升結構的穩定性。我們剃除在此類調節蛋白中具有高度保留性的C端DNA 結合區間 (C-terminal DNA-binding domain) ，藉以研究磷酸化對N端接收磷酸化區間 (N-terminal phosphoreceiver domain) 之影響，結果顯示N端區間表現出與全長相似的結構特性，因此我們推論在多聚化 (Oligomerization) 的現象中，N端區間扮演了關鍵的角色。我們藉由核磁共振更進一步地分析了活化態N端區間 (D56EN) 之構形變化，發現有超過百分之二十的胺基酸有明顯的結構改變。因此我們推論出: 在此雙分子系統受到環境的變化後，藉由磷酸化RcsB的N端區間來產生結構改變，此改變造成四聚體的形成進而提升對啟動子的結合能力，因此促進莢膜多醣體之生合成。

Klebsiella pneumoniae, an important pathogen of hospital infections, necessarily produces plenty of capsular polysaccharide (CPS) to prevent phagocytosis from the hosts. The response regulator RcsB is essential for regulating CPS prodution but its regulatory mechanism remains unclear. Here, we cloned wild-type RcsB to characterize the structural and functional properties. To clarify the effect of phosphorylation in RcsB, a constitutive mutant D56E was also constructed. Electrophoretic mobility shift assay reveals that D56E apparently enhanced its binding ability with cps gene promoter, indicating phosphorylation might raise expression level of cps genes. Moreover, our biophysical studies demonstrate that D56E not only triggers formation of tetramer via charge-charge interactions but also enhances thermal stability, which is not found in wild-type RcsB. According to our bioinformatic analysis, N-terminal phosphoreceiver domain plays an important role in oligomerization. Our results show that N-terminal domain exhibits similar biophysical properties with that of full-length RcsB. Interestingly, N-terminal domain of D56E has a quite different HSQC spectrum as compared with that of wild-type RcsB, implying conformation is moderately changed upon phosphorylation. Hence we proposed that the N-terminal domain has to be phosphorylated to alter its structure, thus triggering the formation of oligomers. This oligomer is critical for promoting DNA-binding in regulation of CPS production.

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