Escherichia coli periplasmic zinc-metallopeptidase BepA normally functions by promoting maturation of LptD, a β-barrel outer membrane protein involved in biogenesis of lipopolysaccharides, but degrades it when its membrane assembly is hampered. These processes should be properly regulated to ensure normal biogenesis of LptD, but the underlying mechanism of regulation, however, remains to be elucidated. A recently solved BepA structure has revealed unique features, in particular the active site is buried in the protease domain and conceivably inaccessible for substrate degradation. Additionally, the His-246 residue in the loop region containing helix α9 (α9/H246 loop), which has a potential flexibility and covers the active site, coordinates the zinc ion as the fourth ligand to exclude a catalytic water molecule, thereby suggesting that the crystal structure of BepA represents a latent form. To examine the roles of the α9/H246 loop in the regulation of the BepA activity, we constructed BepA mutants with a His-246 mutation or a deletion of the α9/H246 loop and analyzed their activities in vivo and in vitro. These mutants exhibited an elevated protease activity and, unlike the wild-type BepA, degraded LptD that is in the normal assembly pathway. In contrast, tethering of the α9/H246 loop repressed the LptD degradation, which suggests that the flexibility of this loop is important to the exhibition of the protease activity. Based on these results, we propose that the α9/H246 loop undergoes a reversible structural change that enables His-246-mediated switching (histidine switch) of its protease activity, which is important for regulated degradation of stalled/misassembled LptD.

中文翻译：

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大肠埃希菌金属肽酶BepA的可逆自抑制调节选择性β桶蛋白降解。

大肠杆菌周质锌金属肽酶BepA通常通过促进LptD的成熟而发挥作用，LptD是参与脂多糖生物合成的一种β桶外膜蛋白，但当其膜组装受到阻碍时，它会降解。这些过程应适当地调节以确保LptD的正常生物发生，但是调节的基本机制尚待阐明。最近解决的BepA结构显示出独特的功能，特别是活性位点被掩埋在蛋白酶域中，并且可能无法进行底物降解。此外，在含有螺旋α9（α9/ H246环）的环区域中的His-246残基具有潜在的柔性并覆盖了活性位点，将锌离子配位为第四配体以排除催化水分子，因此暗示BepA的晶体结构代表潜在形式。为了检查α9/ H246环在BepA活性调节中的作用，我们构建了具有His-246突变或α9/ H246环缺失的BepA突变体，并分析了它们的活性体内和体外。这些突变体表现出升高的蛋白酶活性，并且与野生型BepA不同，其降解了正常装配途径中的LptD。相反，α9/ H246环的束缚抑制了LptD降解，这表明该环的柔韧性对于展现蛋白酶活性很重要。基于这些结果，我们建议α9/ H246环经历可逆的结构变化，使His-246介导的其蛋白酶活性发生转换（组氨酸转换），这对于失速/组装错误的LptD的调控降解很重要。