TY - JOUR

T1 - Evidence for a second regulatory binding site on PspF that is occupied by the C-terminal domain of PspA

AU - Heidrich, Eyleen Sabine

AU - Brüser, Thomas

N1 - Funding Information: This study was funded by the Deutsche Forschungsgemeinschaft (www.dfg.de) by grant BR2285/4-2 of TB. The publication of this article was funded by the Open Access fund of Leibniz Universität Hannover. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Nane Griem-Krey for construction of vector pABS-H10mhip-pspA(145–222), and Inge Reupke and Sybille Traupe for technical assistance. This work was funded by the German Research Foundation (DFG grant BR2285/4-2).

PY - 2018/6/15

Y1 - 2018/6/15

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85055114752&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0198564

DO - 10.1371/journal.pone.0198564

M3 - Article

C2 - 29906279

AN - SCOPUS:85055114752

VL - 13

JO - PLOS ONE

JF - PLOS ONE

SN - 1932-6203

IS - 6

M1 - e0198564

ER -