Structural and Functional Analysis of the C-terminal DNA Binding Domain of the Salmonella typhimurium SPI-2 Response Regulator SsrB
Structural and Functional Analysis of the C-terminal DNA Binding Domain of the Salmonella typhimurium SPI-2 Response Regulator SsrB
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DOI：
10.1074/jbc.m806261200
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发表时间：
2009-05-01
影响因子：
4.8
通讯作者：
Kenney, Linda J.
中科院分区：
文献类型：
--
作者：
Carroll, Ronan K.;Liao, Xiubei;Kenney, Linda J.
在细菌致病过程中，毒力基因的调控受双组分调控系统控制。在大肠杆菌中，EnvZ/OmpR双组分系统被最好地理解为调节外膜蛋白的表达，但是在肠道沙门氏菌中，OmpR激活位于沙门氏菌致病岛2（SPI-2）上的SsrA/B双组分系统的转录。反应调节剂SsrB控制III型分泌系统的表达，其中效应物修饰液泡膜并通过内吞途径防止其降解。沙门氏菌的极性修饰使其能够在巨噬细胞吞噬体中存活和复制，并传播到肝脏和脾脏，引起全身感染。激活EnvZ和SsrA的信号是未知的，但与液泡中遇到的酸性pH值有关。我们以前的工作确定，SsrB结合到AT丰富的DNA区域，序列保守性差。虽然SsrB是沙门氏菌的主要毒力调节因子，但关于它如何结合DNA并激活转录的知之甚少。本研究利用核磁共振技术解析了SsrB的C端DNA结合结构域（SsrBC）的结构，并分析了氨基酸取代对功能的影响。我们鉴定了DNA识别螺旋（Lys（179），Met（186））和二聚化界面（瓦尔（197），Leu（201））中对SsrB转录激活和DNA结合重要的残基。还鉴定了N-末端受体结构域中的必需半胱氨酸残基（Cys（45）），并评价了Cys（203）对二聚化的影响。我们的研究结果表明，虽然二硫键的形成是不需要的二聚化，二聚化发生在DNA结合，并需要随后的转录激活。通过C203 E取代破坏二聚体界面降低SsrB活性。Cys（203）或Cys（45）的修饰可能是SsrB在宿主体内失活的重要方式。
In bacterial pathogenesis, virulence gene regulation is controlled by two-component regulatory systems. In Escherichia coli, the EnvZ/OmpR two-component system is best understood as regulating expression of outer membrane proteins, but in Salmonella enterica, OmpR activates transcription of the SsrA/B two-component system located on Salmonella pathogenicity island 2 (SPI-2). The response regulator SsrB controls expression of a type III secretory system in which effectors modify the vacuolar membrane and prevent its degradation via the endocytic pathway. Vacuolar modification enables Salmonella to survive and replicate in the macrophage phagosome and disseminate to the liver and spleen to cause systemic infection. The signals that activate EnvZ and SsrA are unknown but are related to the acidic pH encountered in the vacuole. Our previous work established that SsrB binds to regions of DNA that are AT-rich, with poor sequence conservation. Although SsrB is a major virulence regulator in Salmonella, very little is known regarding how it binds DNA and activates transcription. In the present work, we solved the structure of the C-terminal DNA binding domain of SsrB (SsrBC) by NMR and analyzed the effect of amino acid substitutions on function. We identified residues in the DNA recognition helix (Lys(179), Met(186)) and the dimerization interface (Val(197), Leu(201)) that are important for SsrB transcriptional activation and DNA binding. An essential cysteine residue in the N-terminal receiver domain was also identified (Cys(45)), and the effect of Cys(203) on dimerization was evaluated. Our results suggest that although disulfide bond formation is not required for dimerization, dimerization occurs upon DNA binding and is required for subsequent activation of transcription. Disruption of the dimer interface by a C203E substitution reduces SsrB activity. Modification of Cys(203) or Cys(45) may be an important mode of SsrB inactivation inside the host.