Figaj Donata, Gieldon Artur, Bartczak Marlena, Koper Tomasz, Zarzecka Urszula, Lesner Adam, Lipinska Barbara, Skorko-Glonek Joanna
Department of General and Medical Biochemistry, Faculty of Biology, University of Gdansk, Poland.
Department of Theoretical Chemistry, Faculty of Chemistry, University of Gdansk, Poland.
FEBS J. 2016 Sep;283(18):3471-87. doi: 10.1111/febs.13822. Epub 2016 Aug 18.
High-temperature requirement A (HtrA; DegP) from Escherichia coli, an important element of the extracytoplasmic protein quality-control system, is a member of the evolutionarily conserved family of serine proteases. The characteristic feature of this protein is its allosteric mode of activation. The regulatory loops, L3, L2, L1 and LD, play a crucial role in the transmission of the allosteric signal. Yet, the role of LD has not been fully elucidated. Therefore, we undertook a study to explain the role of the individual LD residues in inducing and maintaining the proteolytic activity of HtrA. We investigated the influence of amino acid substitutions located within the LD loop on the kinetics of a model substrate cleavage as well as on the dynamics of the oligomeric structure of HtrA. We found that the mutations that were expected to disturb the loop's structure and/or interactions with the remaining regulatory loops severely diminished the proteolytic activity of HtrA. The opposite effect, that is, increased activity, was observed for G174S substitution, which was predicted to strengthen the interactions mediated by LD. HtrAG174S protein had an equilibrium shifted toward the active enzyme and formed preferentially high-order oligomeric forms.
来自大肠杆菌的高温需求A（HtrA；DegP）是胞外蛋白质质量控制系统的一个重要元件，是丝氨酸蛋白酶进化保守家族的成员。该蛋白质的特征是其变构激活模式。调节环L3、L2、L1和LD在变构信号的传递中起关键作用。然而，LD的作用尚未完全阐明。因此，我们开展了一项研究来解释LD单个残基在诱导和维持HtrA蛋白水解活性中的作用。我们研究了位于LD环内的氨基酸取代对模型底物切割动力学以及HtrA寡聚体结构动力学的影响。我们发现，预期会干扰该环结构和/或与其余调节环相互作用的突变会严重降低HtrA的蛋白水解活性。对于G174S取代，观察到了相反的效果，即活性增加，这预计会加强由LD介导的相互作用。HtrAG174S蛋白的平衡向活性酶方向移动，并优先形成高阶寡聚体形式。
