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標題: 核苷酸對於KtrA八聚體環的寡聚體化及熱穩定之影響
The effects of nucleotides on oligomerization and thermostability of KtrA octameric ring
作者: 李振權
Chen-Chung Lee
關鍵字: 鉀離子通道蛋白;KtrAB;c-di-AMP;KtrAB;RCK,;c-di-AMP
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KtrAB複合蛋白在枯草芽孢桿菌是個鉀離子通蛋白,能夠調控鉀攝取離子進入細胞進而調節滲透壓。KtrAB 複合蛋白是由細胞膜上鉀離子通道蛋白KtrB以及在胞質的調控蛋白KtrA所組成。KtrA 屬於Regulator of Conductance of K+ (RCK) 家族之一,其結構為八聚體環。有研究指出當KtrA的RCK N-terminal (RCK_N) domain結合ATP或是ADP後,KtrA會因此發生改變構型,進而調控KtrB離子通道的開關(gating)。先前研究發現新型二級訊號分子c-di-AMP,能夠結合在KtrA 的RCK C-terminal (RCK_C) domain。然而c-di-AMP對於KtrAB複合蛋白的作用機制尚未明確,因此在本研究我們試著釐清c-di-AMP及其他核苷酸對於KtrA的構型及寡聚體的影響。從我們解出的晶體結構發現KtrA RCK_N domain結合ATP似乎阻礙RCK_C domain結合c-di-AMP,因此我們認為先前純化的KtrA沒有經過大量透析以確保沒有內源性的ATP或是ADP結合,導致晶體結構無法看到c-di-AMP。我們透過等溫滴定量熱法 (ITC)和差異性掃描螢光法 (DSF)探討核苷酸與KtrA的結合,並證明KtrA排除ATP殘留後能與c-di-AMP及其他核苷酸結合。從DSF結果中,我們意外發現KtrA結合ATP後造成Tm值下降,但透過動態光散射(DLS)結果看到KtrA仍然上八聚體。過去文獻曾報導c-di-AMP結合KtrA會抑制KtrB的功能,因此我們因此懷疑是否c-di-AMP會造成KtrA八聚體構型改變或瓦解,因此我們利用native gel以及負染電子顯微鏡觀察有無添加c-di-AMP對於KtrA之寡聚體程度。結果發現KtrA結合ci-di-AMP並未造成八聚體瓦解,但因為解析度的限制目前無法得知KtrA八聚體環細微構型變化。未來我們將嘗試使用apo KtrA與c-di-AMP共結晶,並透過X射線晶體學和冷凍電子顯微鏡(cryoEM)得到KtrAB複合蛋白與c-di-AMP結合的高解析度結構。

In Bacillus subtilis, KtrAB complex is a potassium channel which can mediate the osmotic stress by controlling uptake of potassium ions (K+) into cells. KtrAB complex is composed of the potassium channel KtrB, and the cytoplasmic regulator KtrA. KtrA belongs to a Regulator of Conductance of K+ (RCK) family and forms an octameric ring. Structural and functional studies have shown that KtrA adopts different conformations while binding ATP and ADP at the RCK N-terminal (RCK_N) domain, by which mediating the gating of KtrB. Recent studies suggested that c-di-AMP, a new second messenger, can bind to KtrA RCK C-terminal (RCK_C) domain dimer interface. However, the functional effects of c-di-AMP on KtrAB complex remains unclear. In the project, we intend to clarify the conformational and/or oligomerizational impacts of c-di-AMP and nucleotides on KtrA. Previously we determined a crystal structure of KtrA indicating c-di-AMP-binding at RCK_C domain is hindered by ATP-binding at RCK_N domain. It is assumed that endogenous ATP was not removed by extensive dialysis. We utilized Isothermal titration calorimetry (ITC) and Differential Scanning Fluorimetry (DSF) to characterize the interactions between KtrA and nucleotides, indicating a significant binding of c-di-AMP after removal of ATP. Surprisingly, DSF results suggested that the melting temperature (Tm) of KtrA became lower while binding with ATP. However, dynamic light scattering (DLS) results revealed that KtrA remains octameric state in the presence of ATP. As shown in a previous study that KtrA binding to c-di-AMP would inhibit the channel activity of KtrB. Therefore, we speculated that whether c-di-AMP would change the octameric assembly or conformation. We performed native gel electrophoresis and negative stain electron microscope to study the oligomeric state of KtrA. The results demonstrated that KtrA remains its octameric state in the presence of c-di-AMP, whereas the subtle change in octameric assembly is still vague due to the resolution limit. We are currently preparing crystallization trials of ATP-free KtrA with c-di-AMP. The high-resolution structure of c-di-AMP-bound KtrAB complex will also be attempted using X-ray crystallography and cryoEM.
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