Overall, a clear proof of direct protein-protein interaction between CpxA and CpxP is still missing. Here, we show physical interaction between CpxA and CpxP using a bacterial two-hybrid assay. In an alternative approach, membraneStrep-tagged protein interaction experiments were used to demonstrate interaction between CpxP and CpxA in vivo. Additionally, we analyzed the effect of increasing salt concentrations, alkaline pH and misfolded P pilus subunit PapE on the interaction between CpxA and CpxP by mSPINE. Our results show that the interaction between CpxA and CpxP is dynamic and modulated by a high salt concentration and the misfolded pilus subunit PapE. The mechanistic INCB28060 side effects details of signal integration by two-component systems remain incomprehensible. Especially the function of accessory proteins that act as co-sensors is largely unknown. Our results shed light on the interaction between the periplasmic accessory protein CpxP and the sensor kinase CpxA in E. coli. It was known that CpxP does not only inhibit autophosphorylation of CpxA, but is also essential to counteract toxicity of misfolded pilus subunits in collaboration with the DegP protease. For the first time, we demonstrate physical interaction between CpxP and CpxA in unstressed cells. Moreover, using mSPINE we display that this interaction is detached by high NaCl concentration and misfolded pilus subunit PapE. Hence, CpxP modulates CpxA activation by dynamic interaction. Interestingly, increased NaCl concentration does not result in a gradual release, but in full release of CpxP from CpxA in a single step from 250 to 300 mM NaCl. This finding encourages the suggestion that electrostatic interactions might promote the interaction between positively charged residues on the inner surface of CpxP and negatively charged residues on the periplasmic sensor domain of CpxA. The role of electrostatic interactions in protein-protein interactions in general is extensively studied. Thereby, the amount of electrostatic interactions correlates statistically relevant with the binding strength between two proteins and is modulated by the ionic strength of the medium. Moreover, because CpxA autophosphorylation can be induced by salt independently of CpxP in vitro, it is evident that the induced release of CpxP from CpxA by high NaCl concentration is not the only mechanism of the Cpx system to monitor NaCl concentration in the environment. However, the biochemical nature of other mechanisms is so far unclear. In contrast to increasing NaCl concentrations, alkaline pH does not result in the detachment of CpxP from CpxA. This finding specifies a hypothesis of the Raivio group. Biophysical data showed that CpxP “undergoes a subtle structural rearrangement in response to alkaline pH” suggesting that this slight conformational changes might influence the conformation of a binding partner. Moreover, CpxA autophosphorylation can be induced by alkaline pH independently of CpxP.