Signal integration and transmission by the Cpx-TCS of Gram-negative bacteria
Zusammenfassung der Projektergebnisse
Bacteria have developed a variety of mechanisms in order to appropriately encounter extracellular condi¬tions. The ability to correctly sense these conditions via extracellular protein components and transduce such conditions to intracellular domains is essential for bacterial viability. A key function in this context is administered by two-component systems (TCS), which usually encompass a sensor kinase (SK) and a re¬sponse regulator (RR). However, signal integration as well signal transduction within the sensor kinase are still only partly understood. We investigate the E. colt Cpx-TCS (conjugative 2ilus expression) as a model. The Cpx-TCS detects and re¬sponds to perturbations of the cell envelope and consists of the SK CpxA, the RR CpxR, the periplasmic ac¬cessory protein CpxP and the outer membrane lipoprotein NIpE. The key objective of this funding period was the elucidation of TCS-administered extracellular stimuli detection, integration, and the eventual signal transduction across the membrane to the cell interior, in order to generate a specific life-sustaining re¬sponse. The origin of elucidations concerning reaction mechanisms is usually constituted by crystal structures. To date, only the structures of the periplasmic and cytoplasmic parts of CpxA have been successfully solved, which allow first insights into signaling mechanisms. The fact that crystal structures are static and prone to crystallization artefacts requires dynamic investigations for the examination of the accuracy of the respec¬tive crystal structures and derived signaling mechanisms. Electron paramagnetic resonance (EPR) spectros¬copy was applied to study the dynamic inter- and intramolecular structural changes of the sensor kinase CpxA. An important prerequisite was met by establishing the nanodisc technology in the context of the Cpx-TCS, which further allowed the first quantification of protein—protein interactions in the Cpx-TCS. EPR studies eventually demonstrated that by applying CpxP in the absence of ATP, distinct conformational changes on the cytoplasmic side of CpxA occur, which is facilitated by rearranging DHp-domains into a tighter and more rigid conformation. Altogether, these cutting-edge experiments provided first insights into the signal transduction mechanisms of a sensor kinase in context of signaling across the membrane.
