以X-射線結晶學探討汞離子誘發雙重功能轉錄調節因子MerR構型變化之結構機轉

Abstract

汞抗性操作組 (mer operon) 上帶有許多功能性蛋白質基因，可對環境中有毒性的汞離子 (Hg2+) 以及有機汞複合物 (organomercurial compounds) 產生抗性，這些蛋白質具有感應 (sensing protein) 、運輸 (transport protein) 和去毒性 (detoxification protein) 等功能。雙重功能之轉錄調控因子MerR 能對汞抗性操作組之表現與否進行嚴緊的調控。當細菌中沒有汞離子時，MerR 會與汞抗性操作組的轉錄調控區塊 (operator/promoter (O/P)) 結合，抑制汞抗性操作組之轉錄作用，而當汞離子存在時，MerR可與汞離子結合並轉換為轉錄活化因子，啟始汞抗性操作組之轉錄作用，以生成與汞抗性相關之功能性蛋白質。大多數啟動子(promoter)之RNA聚合酶 (RNA polymerase) 結合位-35和-10區塊之間隔為17±1 bps，DNA序列分析發現汞抗性操作組轉錄調控區塊 (mer operon O/P) RNA聚合酶結合位之間隔為19~20 bps，且中間部分帶有一段偽迴文序列 (pseudo-palindromic sequence)，使汞抗性操作組之轉錄調控不同於一般基因之轉錄調控機制。汞抗性操作組轉錄調控區塊與MerR結合後，可經由汞離子引發之MerR二聚體發生構型變化，導致汞抗性操作組轉錄調控區 -10和-35區塊之距離縮短，並旋轉至相同位面上，因而活化轉錄作用。為了瞭解汞離子如何與MerR二聚體結合並活化汞抗性操作組之轉錄，在本實驗中，我們解出了Bacillus megaterium strain MB1之汞抗性操作組轉錄調控因子MerR之原態(apo)與汞離子結合態(Hg2+-bound)兩種狀態下之蛋白質結構(簡稱為apo-MerR和 Hg2+-MerR)，分別代表MerR抑制態 (repressor) 和活化態 (activator)的蛋白質構型。其中apo-MerR結構更是目前已知結構之MerR 家族 (MerR family) 蛋白質中，唯一沒有配體 (ligand) 結合的全長蛋白質構型。比較兩者之蛋白構型差異，不但可以得知MerR二聚體如何組成一對以平面三角配位與汞離子結合的作用位點，並且可了解汞離子與apo-MerR結合之後所引發的二級、三級和四級結構變化。藉由汞離子所引起MerR二聚體之DNA結合位構型變化，可合理解釋MerR與汞離子之交互作用，如何影響轉錄調控區塊之構型變化並調控汞抗性操作組之轉錄作用。

The mer operon confers bacterial resistance to environmental inorganic mercury (Hg2+) and organomercurial compounds by encoding proteins involved in the sensing, transport, and detoxification of these cytotoxic agents. Expression of the mer operon is tightly regulated by the dual-function transcriptional regulatory protein MerR. Whereas in the absence of Hg2+, MerR binds to the operator/promoter region (O/P) of mer operon to supress transcription, MerR is converted into a transcriptional activator upon Hg2+-binding to induce mer operon expression. Sequence analysis suggests that the O/P of mer operon is pseudopalindromic with the -35 and -10 boxes being spaced by 19~20 bps, deviate from the optimal spacing of 17 bp. Therefore, a Hg2+-dependent DNA distortion by the MerR dimer, which brings closer and reorients the two polymerase binding sites, is required to activate transcription. To understand the structural basis by which Hg2+-binding modulates MerR function, we have determined the crystal structures of apo- and Hg2+-bound MerR dimer form Bacillus megaterium MB1, which correspond to the suppressor and activator conformation of MerR, respectively. To our knowledge, the apo-MerR structure represents the first visualization of an inducer-free form of a MerR family protein. Structural comparison not only illustrated how a buried trigonal planar Hg2+-binding pocket is assembled, but also revealed functionally relevant tertiary and quaternary changes between the apo- and Hg2+-bound MerR dimer. The pronounced Hg2+-dependent reposition of the DNA-binding domains suggests a plausible mechanism of transcription regulation by MerR.