Structure and Function of Proteins, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany.
Division of Enteropathogenic Bacteria and Legionella, Robert Koch Institute, 38855 Wernigerode, Germany.
Proc Natl Acad Sci U S A. 2021 Jun 8;118(23). doi: 10.1073/pnas.2017046118.
The virulence factor PlaB promotes lung colonization, tissue destruction, and intracellular replication of , the causative agent of Legionnaires' disease. It is a highly active phospholipase exposed at the bacterial surface and shows an extraordinary activation mechanism by tetramer deoligomerization. To unravel the molecular basis for enzyme activation and localization, we determined the crystal structure of PlaB in its tetrameric form. We found that the tetramer is a dimer of identical dimers, and a monomer consists of an N-terminal α/β-hydrolase domain expanded by two noncanonical two-stranded β-sheets, β-6/β-7 and β-9/β-10. The C-terminal domain reveals a fold displaying a bilobed β-sandwich with a hook structure required for dimer formation and structural complementation of the enzymatic domain in the neighboring monomer. This highlights the dimer as the active form. Δβ-9/β-10 mutants showed a decrease in the tetrameric fraction and altered activity profiles. The variant also revealed restricted binding to membranes resulting in mislocalization and bacterial lysis. Unexpectedly, we observed eight NAD(H) molecules at the dimer/dimer interface, suggesting that these molecules stabilize the tetramer and hence lead to enzyme inactivation. Indeed, addition of NAD(H) increased the fraction of the tetramer and concomitantly reduced activity. Together, these data reveal structural elements and an unprecedented NAD(H)-mediated tetramerization mechanism required for spatial and enzymatic control of a phospholipase virulence factor. The allosteric regulatory process identified here is suited to fine tune PlaB in a way that protects from self-inflicted lysis while ensuring its activity at the pathogen-host interface.
毒力因子 PlaB 促进了军团病病原体的肺部定植、组织破坏和细胞内复制。它是一种高度活跃的磷脂酶，暴露在细菌表面，并通过四聚体解聚寡聚化表现出非凡的激活机制。为了解酶激活和定位的分子基础，我们测定了 PlaB 的四聚体形式的晶体结构。我们发现四聚体是两个相同二聚体的二聚体，单体由 N 端 α/β-水解酶结构域通过两个非典型的两条链 β-折叠 β-6/β-7 和 β-9/β-10 扩展而成。C 端结构域显示出一种折叠结构，呈现出带有钩状结构的双叶 β-夹心结构，该结构对于二聚体形成和相邻单体中酶结构域的结构互补是必需的。这突出了二聚体是活性形式。Δβ-9/β-10 突变体显示出四聚体分数降低和活性谱改变。该变体还显示出对膜的结合受限，导致定位错误和细菌裂解。出乎意料的是，我们在二聚体/二聚体界面观察到了八个 NAD(H)分子，这表明这些分子稳定了四聚体，从而导致酶失活。事实上，添加 NAD(H)增加了四聚体的分数，同时降低了活性。总之，这些数据揭示了结构元件和前所未有的 NAD(H)介导的四聚化机制，这些机制对于磷脂酶毒力因子的空间和酶控制是必需的。这里确定的变构调节过程适合精细调节 PlaB，以保护其免受自身裂解的影响，同时确保其在病原体-宿主界面的活性。
