Purification, reconstitution, and characterization of the CpxRAP envelope stress system of Escherichia coli.
In Escherichia coli the Cpx sensor regulator system senses different kinds of envelope stress and responds by triggering the expression of periplasmic folding factors and proteases. It consists of the membrane-anchored sensor kinase CpxA, the response regulator CpxR, and the periplasmic protein CpxP. The Cpx pathway is induced in vivo by a variety of signals including pH variation, osmotic stress, and misfolded envelope proteins and is inhibited by overproduced CpxP. Because it is not clear how the Cpx pathway is able to recognize and correspond to so many different signals we overproduced, solubilized, purified, and incorporated the complete membrane-integral CpxA protein into proteoliposomes to analyze its biochemical properties in more detail. Autokinase and phosphotransfer activities of the reconstituted CpxA-His6 protein were stimulated by KCl. NaCl also stimulated the activities but to a lesser extent. Other osmotic active solutes as glycine betaine, sucrose, and proline had no effect. The system was further characterized by testing for susceptibility to sensor kinase inhibitors. Among these, Closantel inhibited the activities of solubilized but not of the reconstituted CpxA-His6 protein. We further analyzed the effect of CpxP on CpxA activities. Purified tagless CpxP protein reduced the phosphorylation status of CpxA to 50% but had no effect on CpxA phosphotransfer or phosphatase activities. As the in vitro system excludes the involvement of other factors our finding is the first biochemical evidence for direct protein-protein interaction between the sensor kinase CpxA and the periplasmic protein CpxP resulting in a down-regulation of the autokinase activity of CpxA.
Fleischer
Rebecca
R
Institut für Biologie, Abteilung Physiologie der Mikroorganismen, Humboldt Universität zu Berlin, D-10115 Berlin, Germany.
Heermann
Ralf
R
Jung
Kirsten
K
Hunke
Sabine
S
D001426:Bacterial Proteins / Q000502:physiology*; D001622:Betaine / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000502:physiology*; D000071677:Histidine Kinase; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000737:chemistry; D008565:Membrane Proteins / Q000502:physiology*; D009995:Osmosis; D011189:Potassium Chloride / Q000737:chemistry; D011392:Proline / Q000494:pharmacology; D011489:Protein Denaturation; D017510:Protein Folding; D011494:Protein Kinases / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction; D012965:Sodium Chloride / Q000737:chemistry
D001426:Bacterial Proteins; C491730:CpxP protein, E coli; D029968:Escherichia coli Proteins; D008081:Liposomes; D008565:Membrane Proteins; C085504:CpxR protein, Bacteria; D001622:Betaine; D012965:Sodium Chloride; D011189:Potassium Chloride; D011392:Proline; D011494:Protein Kinases; D000071677:Histidine Kinase; C490475:CpxA protein, E coli