The ClpP serine peptidase is a tetradecameric degradation molecular machine involved in many physiological processes. It becomes a competent ATP-dependent protease when coupled with Clp-ATPases. Small chemical compounds, acyldepsipeptides (ADEPs), are known to cause the dysregulation and activation of ClpP without ATPases and have potential as novel antibiotics. Previously, structural studies of ClpP from various species revealed its structural details, conformational changes, and activation mechanism. Although product release through side exit pores has been proposed, the detailed driving force for product release remains elusive. Herein, we report crystal structures of ClpP from Bacillus subtilis (BsClpP) in unforeseen ADEP-bound states. Cryo-electron microscopy structures of BsClpP revealed various conformational states under different pH conditions. To understand the conformational change required for product release, we investigated the relationship between substrate hydrolysis and the pH-lowering process. The production of hydrolyzed peptides from acidic and basic substrates by proteinase K and BsClpP lowered the pH values. Our data, together with those of previous findings, provide insight into the molecular mechanism of product release by the ClpP self-compartmentalizing protease.

中文翻译：

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通过 pH 下降结合底物水解对 ClpP 蛋白酶侧出口开孔的结构洞察

ClpP 丝氨酸肽酶是一种参与许多生理过程的十四聚体降解分子机器。当与 Clp-ATPases 结合时，它成为一种有效的 ATP 依赖性蛋白酶。已知小型化合物酰基缩肽 (ADEP) 会在没有 ATP 酶的情况下引起 ClpP 的失调和激活，并且具有作为新型抗生素的潜力。以前，来自不同物种的 ClpP 的结构研究揭示了其结构细节、构象变化和激活机制。虽然已经提出通过侧出口孔释放产品，但产品释放的详细驱动力仍然难以捉摸。在此，我们报告了枯草芽孢杆菌ClpP 的晶体结构(BsClpP) 处于不可预见的 ADEP 结合状态。BsClpP 的冷冻电子显微镜结构揭示了不同 pH 条件下的各种构象状态。为了解产品释放所需的构象变化，我们研究了底物水解与 pH 降低过程之间的关系。通过蛋白酶 K 和 BsClpP 从酸性和碱性底物生产水解肽降低了 pH 值。我们的数据与之前的研究结果一起，提供了对 ClpP 自区室化蛋白酶释放产物的分子机制的深入了解。