Ter proteins mediate tellurite resistance and other responses to extracellular stimuli, but their functions are not understood yet. To shed light into the function of KP-TerD, a 20.5 kDa tellurite resistance protein from a plasmid of Klebsiella pneumoniae, we solved its three-dimensional structure in solution by multidimensional NMR spectroscopy. The structure consists of a beta-sandwich formed by two five-stranded beta-sheets and six short helices. Interestingly, the structure contains two clusters of negatively charged residues, which suggested that KP-TerD might bind some metal ions. Using NMR, CD and ICP-OES, we demonstrate that KP-TerD binds two calcium ions. An EDTA competition assay monitored by 1D NMR spectroscopy showed the estimated dissociation constants of KP-TerD. Since the Ca2+ concentration in the cytoplasm of bacteria is estimated to be 90-300 nM, the estimated Kd of site Ca2 of KP-TerD (200 nM) is well suited to sense changes in the cytoplasmic Ca2+ concentrations of bacteria. The calcium ligands are highly conserved in TerD proteins, suggesting that calcium binding is a conserved property of this family. Moreover, the calcium-binding motif of KP-TerD is conserved in TerE and TerZ. Using analogous methodology, we show that KP-TerE and KP-TerZ also bind two calcium ions, but their affinties are different from KP-TerD. These results show that three out of seven Ter proteins bind calcium and their affinities cover a wide range (from □M to nM) that is ideal to respond to changes in cytoplasmic calcium concentration. Overall, these data suggest that TerD, TerE and TerZ function as calcium sensors and that some form of calcium signaling is critical for the Ter response to extracellular stimuli.

中文摘要
克雷伯氏肺炎桿菌屬於腸道革蘭氏陰性桿菌G (-)，會對醫院內住院，衰弱或免疫功能低下的病人造成感染。碲酸鹽化合物(TeO32-)早期被應用於治療由結核分枝桿菌引起的傳染病，並且廣泛的被應用在工業界。目前為止已有數個碲酸鹽抗藥性基因早就被分離鑑定出來，但是對於這些基因如何調控不同的細胞外刺激，例如對噬菌體(phage)、大腸桿菌素(colicin)、碲酸鹽類存在威脅時所做出反應的機制仍然是未知的。亞碲酸鉀(potassium tellurite)可以抑制大多數革蘭氏陰性菌的生長，但是克雷伯氏菌可以在含有亞碲酸鉀的環境下生長。為了闡明克雷伯氏肺炎桿菌質粒(plasmid)中的KP- TerD，20.5 kDa的碲酸鹽抗藥性蛋白的功能，我們運用核磁共振光譜的方法解出其三維立體結構。KP- TerD的蛋白質結構包含兩邊各由五個β-摺版所形成的類似三明治夾層及六個短的螺旋構型。在結構的上方顯示有兩個負電聚集的區域，這表示KP- TerD可能會與帶正電的金屬離子結合。接著我們運用了圓二色譜(circular dichroism)、核磁共振(NMR)以及感應耦合電漿-光學放射光譜(ICP-OES)實驗證實KP-TerD可與鈣離子以1:2結合比例結合，且鈣離子結合的區域的確是在KP-TerD的上方負電聚集的區域。TerD蛋白質家族有獨特的蛋白折疊法和新型的鈣離子結合模式。用EDTA競爭的方法，再藉由一維核磁共振光譜測量，可得到KP- TerD的鈣離子解離常數的估計值，已知在細菌細胞質內的鈣離子濃度估計值為90-300 nM，而KP- TerD之第二個鈣離子的解離常數估計值200 nM是非常適合用來感應在細菌細胞質內鈣離子濃度的變化。與鈣離子結合的配體在不同種類的細菌內的TerD蛋白質是非常一致的，這使人聯想到與鈣離子結合是的TerD蛋白質家族一致的特性。此外，KP- TerD鈣離子結合的區塊也一致的存在於TerE和TerZ蛋白質中。使用類似的方法，我們證明KP-TerE和KP-TerZ也結合兩個鈣離子，但他們的對鈣離子的親和力與KP- TerD不同。這些結果顯示，在七個碲酸鹽抗藥性蛋白質之中有三個會與鈣離子結合，且其與鈣離子的結合親合力涵蓋的範圍很廣（從□M到nM），對於細胞漿內的鈣離子濃度變化可做出理想的回應。總體而言，這些實驗結果推測出TerD，TerE和TerZ這三個蛋白質的功能可能是做為鈣離子傳導感應器，並且可能是碲酸鹽抗藥性以及細胞外刺激所引起的某種形式的鈣離子訊息傳導重要的關鍵。

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