Dimerization of the quorum-sensing transcription factor TraR enhances resistance to cytoplasmic proteolysis.
Dimerization of the quorum-sensing transcription factor TraR enhances resistance to cytoplasmic proteolysis.
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DOI：
10.1111/j.1365-2958.2009.06730.x
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发表时间：
2009-07
影响因子：
3.6
通讯作者：
Winans SC
中科院分区：
文献类型：
--
作者：
Pinto UM;Winans SC
TRAR是一种由根癌农杆菌Ti质粒编码的LuxR型群体感应蛋白。TRIR的生物活性需要信息素3-氧辛酰高丝氨酸内酯(OOHL)，在溶液中和与DNA结合时都是二聚体。二聚化主要由两个α螺旋介导，一个在N端的OOH1结合域，另一个在C端的DNA结合域。这些螺旋中的每一个都形成一个平行的螺旋线圈，具有相反亚基的相同螺旋。我们以前已经证明Oohl对于抵抗蛋白分解是必不可少的，这里我们询问二聚化是否也是蛋白酶抵抗所必需的。我们在二聚体界面构建了一系列定点突变，并测试了这些突变体在体内的活性。A149、A150、A153、A222和I229残基的改变完全丧失了活性，另外三个残基的改变也造成了明显的缺陷。所有突变体都进行了二聚化和特异性DNA结合测试。这些蛋白质在根癌农杆菌中的细胞丰度是用免疫印迹和Oohl-隔离来测量的，而半衰期是用脉冲追逐放射性标记来测量的。我们发现体内活性缺陷、体外二聚体缺陷、DNA结合缺陷、蛋白质半衰期缺陷之间存在相关性。我们得出结论，TRAR的二聚化增强了对细胞蛋白水解酶的抗性。
TraR is a LuxR-type quorum sensing protein encoded by the Ti plasmid of Agrobacterium tumefaciens. TraR requires the pheromone 3-oxooctanoylhomoserine lactone (OOHL) for biological activity, and is dimeric both in solution and when bound to DNA. Dimerization is mediated primarily by two alpha helices, one in the N-terminal OOHL binding domain, and the other in the C-terminal DNA binding domain. Each of these helices forms a parallel coiled coil with the identical helix of the opposite subunit. We have previously shown that OOHL is essential for resistance to proteolysis, and here we asked whether dimerization is also required for protease resistance. We constructed a series of site-directed mutations at the dimer interface, and tested these mutants for activity in vivo. Alteration of residues A149, A150, A153, A222 and I229 completely abolished activity, while alteration of three other residues also caused significant defects. All mutants were tested for dimerization as well as for specific DNA binding. The cellular abundance of these proteins in A. tumefaciens was measured using western immunoblots and OOHL-sequestration, while the half-life was measured by pulse-chase radiolabelling. We found a correlation between defects in in vivo activity, in vitro dimerization, DNA binding, protein halflife. We conclude that dimerization of TraR enhances resistance to cellular proteases.
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影响因子：
10.5
作者：
Herman, C;Thévenet, D;D'Ari, R
通讯作者：
D'Ari, R
DOI：
10.1073/pnas.0506163102
发表时间：
2005-12-20
影响因子：
11.1
作者：
Shi, K;Brown, CK;Earhart, CA
通讯作者：
Earhart, CA
影响因子：
3.6
作者：
Chai, Y;Zhu, J;Winans, SC
通讯作者：
Winans, SC
影响因子：
56.9
作者：
SCHULTZ, SC;SHIELDS, GC;STEITZ, TA
通讯作者：
STEITZ, TA
影响因子：
3.2
作者：
EBERHARD, A
通讯作者：
EBERHARD, A