Gloeckl Sarina, Tyndall Joel D A, Stansfield Scott H, Timms Peter, Huston Wilhelmina M
Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Qld., Australia.
J Mol Microbiol Biotechnol. 2012;22(1):10-6. doi: 10.1159/000336312. Epub 2012 Feb 21.
HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.
HtrA是一种复杂的多聚体伴侣蛋白和丝氨酸蛋白酶，对许多细菌的毒力和生存至关重要。沙眼衣原体是一种专性胞内细菌病原体，可导致严重的疾病病理。沙眼衣原体HtrA（CtHtrA）已被证明在疾病的实验室模型中高度表达。在本研究中，CtHtrA蛋白活性位点结构的分子建模确定了推定的S1 - S3亚位点残基I242、I265和V266。这些残基通过定点诱变进行了改变，结果表明这些变化显著降低了对已知底物的蛋白酶活性，并且与野生型相比，底物范围更窄且更具特异性。细菌双杂交分析表明，CtHtrA能够在体内与广泛的蛋白质序列以高亲和力相互作用。然而，值得注意的是，当残基V266发生突变时，69个克隆中有35个的相互作用发生了显著改变，表明该残基在底物结合过程中具有重要功能。
