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膜应力反应系统的关键组成部分。PspA的低聚和聚集特性阻碍了其的生化和功能表征。这是最近可以解决一个完全可溶PSPA片段的卷曲螺旋结构，PSPA（1-144），其中σ缔合⁵⁴增强子结合蛋白PspF在其W56环处，从而下调Psp反应。现在我们发现，在没有全长PspA的情况下，PspA的C末端部分PspA（145–222）也可与PspF相互作用并抑制其活性。令人惊讶的是，在存在全长PspA的情况下，PspA（145–222）的作用完全改变，因为启动子活性被触发而不是在此条件下被抑制。因此，PspA（145–222）干扰了全长PspA对PspF的抑制作用，很可能是通过与仍与PspF结合的全长PspA相互作用而产生的。为了支持这种观点，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PspA / PspA和PspA / PspF相互作用的第一个直接演示在体内由PspA的C端介导。数据表明，PspF上的调节结合位点不仅存在于PspA的N末端卷曲螺旋结构域，而且还存在于C末端结构域。PspA-（145–222）对PspF的抑制作用在膜应力作用下有所降低，而PspA（1–144）对PspF的抑制作用对膜应力无反应。因此，我们建议PspA的C末端域对于响应Psp系统刺激而调节PspF至关重要。