Cage assembly of DegP protease is not required for substrate-dependent regulation of proteolytic activity or high-temperature cell survival
Cage assembly of DegP protease is not required for substrate-dependent regulation of proteolytic activity or high-temperature cell survival
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DOI：
10.1073/pnas.1204791109
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发表时间：
2012-05-08
影响因子：
11.1
通讯作者：
Sauer, Robert T.
中科院分区：
文献类型：
--
作者：
Kim, Seokhee;Sauer, Robert T.
DegP是高度保守的HtrA家族的成员，负责对革兰氏阴性细菌周质中错误折叠的蛋白质进行质量控制降解，是大肠杆菌高温生存所必需的。底物结合将DegP从包含两个三聚体的非活性低聚物转变为活性多面体笼子，通常包含四个或八个三聚体。尽管这些观察表明两者之间存在因果关系，但我们发现CAGE组装和蛋白水解酶的激活是可以分离的。事实上，在有或没有底物的情况下保持三聚体、六聚体或十二聚体的DegP变体在体外仍然表现出强劲的和积极的合作底物降解，最重要的是，维持高温细菌的生长以及野生型酶。我们的结果支持一个模型，在该模型中，底物结合将不活跃的三聚体转化为具有蛋白水解性的三聚体，同时通过加强一个三聚体中的PDZ1结构域与相邻三聚体中的PDZ2‘结构域的结合而导致笼状组装。因此，这两个过程都依赖于底物结合，但它们可以在不损失生物学功能的情况下解偶联。我们讨论了潜在的偶联机制，以及为什么笼的形成可能已经进化，如果它不是DegP蛋白降解所必需的。
DegP, a member of the highly conserved HtrA family, performs quality-control degradation of misfolded proteins in the periplasm of Gram-negative bacteria and is required for high-temperature survival of Escherichia coli. Substrate binding transforms DegP from an inactive oligomer containing two trimers into active polyhedral cages, typically containing four or eight trimers. Although these observations suggest a causal connection, we show that cage assembly and proteolytic activation can be uncoupled. Indeed, DegP variants that remain trimeric, hexameric, or dodecameric in the presence or absence of substrate still display robust and positively cooperative substrate degradation in vitro and, most importantly, sustain high-temperature bacterial growth as well as the wild-type enzyme. Our results support a model in which substrate binding converts inactive trimers into proteolytically active trimers, and simultaneously leads to cage assembly by enhancing binding of PDZ1 domains in one trimer to PDZ2' domains in neighboring trimers. Thus, both processes depend on substrate binding, but they can be uncoupled without loss of biological function. We discuss potential coupling mechanisms and why cage formation may have evolved if it is not required for DegP proteolysis.