Institute of Health and Biomedical Innovation (IHBI), Queensland University of Technology (QUT), 60 Musk Avenue, Kelvin Grove, Queensland, Australia, 4059.
Cell Mol Biol Lett. 2013 Dec;18(4):522-37. doi: 10.2478/s11658-013-0103-2. Epub 2013 Sep 13.
Chlamydia trachomatis is a bacterial pathogen responsible for one of the most prevalent sexually transmitted infections worldwide. Its unique development cycle has limited our understanding of its pathogenic mechanisms. However, CtHtrA has recently been identified as a potential C. trachomatis virulence factor. CtHtrA is a tightly regulated quality control protein with a monomeric structural unit comprised of a chymotrypsin-like protease domain and two PDZ domains. Activation of proteolytic activity relies on the C-terminus of the substrate allosterically binding to the PDZ1 domain, which triggers subsequent conformational change and oligomerization of the protein into 24-mers enabling proteolysis. This activation is mediated by a cascade of precise structural arrangements, but the specific CtHtrA residues and structural elements required to facilitate activation are unknown. Using in vitro analysis guided by homology modeling, we show that the mutation of residues Arg362 and Arg224, predicted to disrupt the interaction between the CtHtrA PDZ1 domain and loop L3, and between loop L3 and loop LD, respectively, are critical for the activation of proteolytic activity. We also demonstrate that mutation to residues Arg299 and Lys160, predicted to disrupt PDZ1 domain interactions with protease loop LC and strand β5, are also able to influence proteolysis, implying their involvement in the CtHtrA mechanism of activation. This is the first investigation of protease loop LC and strand β5 with respect to their potential interactions with the PDZ1 domain. Given their high level of conservation in bacterial HtrA, these structural elements may be equally significant in the activation mechanism of DegP and other HtrA family members.
沙眼衣原体是一种细菌病原体，导致了全球最普遍的性传播感染之一。它独特的发育周期限制了我们对其致病机制的理解。然而，CtHtrA 最近被确定为一种潜在的沙眼衣原体毒力因子。CtHtrA 是一种受严格调控的质量控制蛋白，其单体结构单元由糜蛋白酶样蛋白酶结构域和两个 PDZ 结构域组成。蛋白水解活性的激活依赖于底物的 C 端通过别构结合到 PDZ1 结构域，从而触发随后的构象变化和蛋白质寡聚化形成 24 聚体以进行蛋白水解。这种激活是由一系列精确的结构排列介导的，但促进激活所需的特定 CtHtrA 残基和结构元件尚不清楚。我们通过同源建模指导的体外分析表明，突变预测会破坏 CtHtrA PDZ1 结构域与环 L3 之间以及环 L3 与环 LD 之间相互作用的残基 Arg362 和 Arg224 对于蛋白水解活性的激活至关重要。我们还证明，突变预测会破坏 PDZ1 结构域与蛋白酶环 LC 和β5 链之间相互作用的残基 Arg299 和 Lys160 也能够影响蛋白水解，这表明它们参与了 CtHtrA 的激活机制。这是首次针对蛋白酶环 LC 和β5 进行的关于它们与 PDZ1 结构域潜在相互作用的研究。鉴于它们在细菌 HtrA 中高度保守，这些结构元件在 DegP 和其他 HtrA 家族成员的激活机制中可能同样重要。
