Structure and functional properties of the Bacillus subtilis transcriptional repressor Rex
Structure and functional properties of the Bacillus subtilis transcriptional repressor Rex
复制标题
DOI：
10.1111/j.1365-2958.2008.06295.x
复制
发表时间：
2008-07-01
影响因子：
3.6
通讯作者：
von Wachenfeldt, Claes
中科院分区：
文献类型：
--
作者：
Wang, Ellen;Bauer, Mikael C.;von Wachenfeldt, Claes
转录因子Rex在几种革兰氏阳性细菌中参与了对发酵生长和低氧条件下生长重要基因的表达调控。Rex通过NADH/NAD（+）比值的变化来感知细胞的氧化还原平衡。来自水热菌和嗜热菌的两种基本相同的Rex蛋白的晶体结构先前已与NADH复合物确定。在这里，我们介绍了枯草芽孢杆菌的Rex蛋白的晶体结构，以及它对核苷酸和DNA的亲和力的广泛研究，使用表面等离子体共振，等温滴定量热法和电泳迁移率转移测定。我们发现Rex对NADH有很高的亲和力，但对NAD（+）的亲和力低2万倍。然而，DNA结合后，NAD（+）的亲和力增加了30倍，这表明DNA结合与NAD（+）结合之间存在正变构耦合。两种伪载子形式的晶体结构（来自NADH浸泡和ATP共结晶的晶体）显示出与先前确定的Rex:NADH配合物非常不同的构象，其中n端结构域远离二聚体核心。提出了一种机制，即c端结构域交换螺旋的构象变化介导了从柔性DNA结合形式到无法结合DNA的锁定nadh结合形式的转变。
The transcription factor Rex has been implicated in regulation of the expression of genes important for fermentative growth and for growth under conditions of low oxygen tension in several Gram-positive bacteria. Rex senses the redox poise of the cell through changes in the NADH/NAD(+) ratio. The crystal structures of two essentially identical Rex proteins, from Thermus aquaticus and T. thermophilus, have previously been determined in complex with NADH. Here we present the crystal structure of the Rex protein from Bacillus subtilis, as well as extensive studies of its affinity for nucleotides and DNA, using surface plasmon resonance, isothermal titration calorimetry and electrophoretic mobility shift assays. We show that Rex has a very high affinity for NADH but that its affinity for NAD(+) is 20 000 times lower. However, the NAD(+) affinity is increased by a factor of 30 upon DNA binding, suggesting that there is a positive allosteric coupling between DNA binding and NAD(+) binding. The crystal structures of two pseudo-apo forms (from crystals soaked with NADH and cocrystallized with ATP) show a very different conformation from the previously determined Rex:NADH complexes, in which the N-terminal domains are splayed away from the dimer core. A mechanism is proposed whereby conformational changes in a C-terminal domain-swapped helix mediate the transition from a flexible DNA binding form to a locked NADH-bound form incapable of binding DNA.