From the Institute of Microbiology, Leibniz Universität Hannover, Hannover, Germany.
PLoS One. 2018 Jun 15;13(6):e0198564. doi: 10.1371/journal.pone.0198564. eCollection 2018.
PspA is a key component of the bacterial Psp membrane-stress response system. The biochemical and functional characterization of PspA is impeded by its oligomerization and aggregation properties. It was recently possible to solve the coiled coil structure of a completely soluble PspA fragment, PspA(1-144), that associates with the σ54 enhancer binding protein PspF at its W56-loop and thereby down-regulates the Psp response. We now found that the C-terminal part of PspA, PspA(145-222), also interacts with PspF and inhibits its activity in the absence of full-length PspA. Surprisingly, PspA(145-222) effects changed completely in the presence of full-length PspA, as promoter activity was triggered instead of being inhibited under this condition. PspA(145-222) thus interfered with the inhibitory effect of full-length PspA on PspF, most likely by interacting with full-length PspA that remained bound to PspF. In support of this view, a comprehensive bacterial-2-hybrid screen as well as co-purification analyses indicated a self-interaction of PspA(145-222) and an interaction with full-length PspA. This is the first direct demonstration of PspA/PspA and PspA/PspF interactions in vivo that are mediated by the C-terminus of PspA. The data indicate that regulatory binding sites on PspF do not only exist for the N-terminal coiled coil domain but also for the C-terminal domain of PspA. The inhibition of PspF by PspA-(145-222) was reduced upon membrane stress, whereas the inhibition of PspF by PspA(1-144) did not respond to membrane stress. We therefore propose that the C-terminal domain of PspA is crucial for the regulation of PspF in response to Psp system stimuli.
PspA 是细菌 Psp 膜应激反应系统的关键组成部分。由于 PspA 的寡聚化和聚集特性，其生化和功能特性的表征受到阻碍。最近，人们有可能解决完全可溶性 PspA 片段 PspA(1-144)的卷曲螺旋结构，该片段与 σ54 增强子结合蛋白 PspF 结合，在其 W56 环处，从而下调 Psp 反应。我们现在发现 PspA 的 C 端部分 PspA(145-222)也与 PspF 相互作用，并在没有全长 PspA 的情况下抑制其活性。令人惊讶的是，在全长 PspA 存在的情况下，PspA(145-222)的作用完全改变，因为在这种条件下，启动子活性被触发而不是被抑制。因此，PspA(145-222)通过与仍与 PspF 结合的全长 PspA 相互作用，干扰全长 PspA 对 PspF 的抑制作用。支持这一观点的是，全面的细菌-2 杂交筛选以及共纯化分析表明 PspA(145-222)的自我相互作用以及与全长 PspA 的相互作用。这是首次直接证明 PspA/PspA 和 PspA/PspF 相互作用在体内是由 PspA 的 C 端介导的。数据表明，PspF 上的调节结合位点不仅存在于 N 端卷曲螺旋结构域，也存在于 PspA 的 C 端结构域。PspA-(145-222)对 PspF 的抑制作用在膜应激时降低，而 PspA(1-144)对 PspF 的抑制作用对膜应激没有反应。因此，我们提出 PspA 的 C 端结构域对于 PspF 响应 Psp 系统刺激的调节至关重要。
