雙分子系統是細菌中最常用來感測外在環境訊號並傳遞到細胞內的主要裝置。PmrAB雙分子訊息傳遞系統主要用來感測外在環境例如低鎂離子與弱酸條件，接著在病原體內調控與脂多醣修飾及多黏菌素B的抗藥性相關基因。在沙門氏桿菌中PmrD小分子鹼性蛋白負責保護Phospho-PmrA蛋白上的磷酸根免於內生性或PmrB去磷酸脢的去磷酸化作用。PmrD是目前所知唯一能連結PmrA/PmrB與PhoP/PhoQ這兩套雙分子系統的鹼性小分子蛋白。首先，本研究發現克雷博氏肺炎桿菌內的PmrD重組蛋白剛開始同時具有氧化態與還原態的構造，但在兩星期內會在空氣中自動轉變成完全氧化態。原二色旋光光譜儀顯示這兩種構型的二級結構與Tm值有些微的不同。質譜跟核磁共振光譜分析確認在氧化態蛋白中的Cys17與Cys35會形成一對雙硫鍵。同時化學位移分析也顯示氧化態與還原態PmrD蛋白有很大改變，進一步顯示他們的結構差異性。仔細分析這兩種蛋白的二級結構發現主要構型差異處位於蛋白質C-端的□螺旋。PmrD蛋白同時具有氧化與還原態的特性之前未曾被發現，而體內多黏菌素B抗藥性測試則顯示在形成雙硫鍵處的Cys17與Cys35產生突變會造成抗藥性降低，表示他們與克雷博氏肺炎桿菌的抗性功能有相關。接著，我們解出與沙門氏桿菌功能相似的克雷博氏肺炎桿菌PmrD蛋白(KP-PmrD)結構，並與大腸桿菌PmrD蛋白比較，結構與骨架動力學數據結果顯示KP-PmrD的C端□螺旋較短且更具有可動性。為了進一步瞭解KP-PmrD與PmrA N端訊息接收區(PmrAN)之間的分子交互作用，我們使用小分子磷酸化模擬物BeF3-進行核磁共振與表面電漿共振的定性與定量分析。結果顯示在BeF3-存在下KP-PmrD會與PmrAN進行專一性反應（KD=1.74±0.81 □M）。當與BeF3-活化後的PmrAN結合時，化學位移擾動與交叉飽和實驗可定義出KP-PmrD中由□-桶狀結構loops 2,4,6上所組成的一塊連續氨基酸群(Trp3, Leu26, Met28, Asp50, Ala51, and Ile65)。最後，雖然許多反應調節蛋白的結構已經被解出，但關於該蛋白與PmrD連結蛋白的複合體結構仍不是很清楚，之前我們已經決定克雷博氏肺炎桿菌PmrD蛋白的結構及其與磷酸化PmrA N端訊息接收區(PmrAN)的辨識模式，這裡我們使用資料驅動分子嵌合程式HADDOCK與定點電子自旋標記方法提出一種作用模式，在該模式中PmrAN透過活性部位區和KP-PmrD發生作用，而它也是一般反應調節蛋白常用來與組胺酸激酶進行分子間蛋白質交互作用的區域。這些結果顯示，定點電子自旋標記方法可作為另種驗證由HADDOCK所得之KP-PmrD/PmrAN蛋白複合體結構之生物物理方式並提供蛋白質間之長距離結構資訊。本研究針對克雷博氏肺炎桿菌PmrAB雙分子系統中PmrD蛋白對於多黏菌素B的調控提供更深入的瞭解。且我們的結果應可對KP-PmrD蛋白與PmrAB雙分子系統中Phospho-PmrA的專一性反應提供以結構為基礎的洞見。

In bacteria, the two-component signal-transduction system is the most common system for sensing environmental signals and transducing the information into the cell. The PmrA-PmrB two-component signal transduction system, responsible for sensing external stimuli of low Mg2+ and mild acidic conditions, can control the genes involved in lipopolysaccharide (LPS) modification and polymyxin resistance in pathogens. The small basic protein PmrD, a polymyxin B resistance protein, is the only protein known to be able to connect PmrA/PmrB and PhoP/PhoQ two-component systems. First, we found that the recombinant PmrD of Klebsiella penumoniae (KP-PmrD) initially contains both oxidized and reduced forms, and the reduced form is nearly converted into the oxidized form within two weeks in the air. The CD spectra revealed the secondary structure and the melting temperature are somewhat different between the two forms. Both mass analysis and NMR data confirmed the formation of a disulfide bridge at Cys17 and Cys35 for the oxidized form. Dramatic chemical-shift changes between the two forms were observed, further revealing their structural difference. A detailed comparison of secondary structures between the two forms showed that the major conformational difference is located at C-terminal □-helix. The existence of both reduced and oxidized forms has not been previously reported for any PmrD, and in vivo polymyxin B susceptibility assay of the site-directed mutagenesis on KP-PmrD reveals that the two cysteine, which form a disulfide bridge between Cys17-Cys35, is functional relevant. Second, we provided the NMR solution structure of PmrD in Klebsiella pneumoniae (KP-PmrD), which showed similar in vitro phosphotransfer activity to Salmonella PmrD. Structural comparisons and backbone dynamics data revealed that the C-terminal □-helix of KP-PmrD is shorter and much more flexible. To further study the molecular interactions involved in KP-PmrD and the N-terminal receiver domain of PmrA (PmrAN), we performed quantitative and qualitative SPR as well as NMR analyses both in the absence and presence of the phosphoryl analog beryllofluoride (BeF3-). The results demonstrated that KP-PmrD participates in specific interactions (KD = 1.74 ± 0.81 □M) with the PmrAN in the presence of BeF3-. Chemical shift perturbations and cross-saturation experiments defined a cluster of residues (Trp3, Leu26, Met28, Asp50, Ala51, and Ile65) forming a contiguous patch near loops 2, 4, and 6 of the □-barrel upon binding with the BeF3--activated PmrAN. Third, while several structures of receiver domain of response regulators have been solved, structures of regulatory domain in complex with its connector protein have remained elusive. We used the data-driven docking method HADDOCK and site-directed spin labeling approaches to proposed an interaction mode in which the N-terminal receiver domain of PmrA (PmrAN) interacts with KP-PmrD through the active site pockets, which is widely used by response regulatory domains for intermolecular protein/protein interactions with the histidine kinase. The results demonstrate that site-directed spin labeling could provide an independent biophysical method to confirm the complex model from HADDOCK and validate long-range distance information between corresponding regions of KP-PmrD/PmrAN complex structure. This study furthers the understanding of KP-PmrD polymyxin B resistance regulation in the PmrAB TCS and our results should provide insights into the structural basis of KP-PmrD for specific interaction with the phospho-PmrA in the PmrAB two-component system.

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