Phosphorylation Alters the Interaction of the Response Regulator OmpR with Its Sensor Kinase EnvZ

Abstract

OmpR and EnvZ comprise a two-component system that regulates the porin genes ompF and ompC in response to changes in osmolarity. EnvZ is autophosphorylated by intracellular ATP on a histidine residue, and it transfers the phosphoryl group to an aspartic acid residue of OmpR. EnvZ can also dephosphorylate phospho-OmpR (OmpR-P) to control the cellular level of OmpR-P. At low osmolarity, OmpR-P levels are low because of either low EnvZ kinase or high EnvZ phosphatase activities. At high osmolarity, OmpR-P is elevated. It has been proposed that EnvZ phosphatase is the activity that is regulated by osmolarity. OmpR is a twodomain response regulator; phosphorylation of OmpR increases its affinity for DNA, and DNA binding stimulates phosphorylation. The step that is affected by DNA depends upon the phosphodonor employed. In the present work, we have used fluorescence anisotropy and phosphotransfer assays to examine OmpR interactions with EnvZ. Our results indicate that phosphorylation greatly reduces the affinity of OmpR for the kinase, whereas DNA does not affect their interaction. The results presented cast serious doubts on the role of the EnvZ phosphatase in response to signaling in vivo.The predominant paradigm for signal transduction in prokaryotes is the two-component regulatory system (see Ref. 1 for reviews and references). The first component is a sensor kinase, often a membrane protein, which senses the appropriate environmental signal and is phosphorylated from intracellular ATP on a histidine residue. The sensor phosphokinase then transfers the phosphoryl group to an aspartic acid residue of the second component, the response regulator. Phosphorylation of the response regulator generally leads to its activation, often by increasing its affinity for DNA with a subsequent effect on transcription. In a postulated second level of regulation, it has been suggested that the sensor kinase can also stimulate dephosphorylation of the phosphorylated response regulator via a phosphatase activity, thus limiting the level of the activated regulator and resetting the system.The two-component regulatory system that governs expression of the outer membrane porins OmpF and OmpC consists of the sensor kinase EnvZ and the response regulator OmpR. Activation of EnvZ by an unknown signal, related to the osmolarity of the growth medium, leads to phosphorylation of OmpR at aspartate 55 (2-4). Phosphorylation of OmpR results in an increased affinity for the regulatory regions upstream from the ompF and ompC genes (5-8). Recent in vitro studies demonstrated that the corollary is also true, i.e. the presence of DNA increases the level of OmpR phosphorylation (9, 10). As a result of these studies, we proposed that OmpR exists as an equilibrium mixture of four distinct states (see Fig. 1): unphosphorylated OmpR (A), phosphorylated OmpR (B), unphosphorylated OmpR bound to DNA with low affinity (C), and phosphorylated OmpR bound to DNA with high affinity (D). A similar model has been proposed for the single-domain response regulator Ch...

The Linker Region Plays an Important Role in the Interdomain Communication of the Response Regulator OmpR

“…GGK also binds DNA, as shown in the footprinting assay below. The interaction of GGK with DNA decreases the stability of GGK-P; this effect is diametrically opposed to that observed for OmpR (24).…”

“…3d, lanes 1-3), but the addition of DNA has a remarkable effect on the level of GGK-P (lanes 4 -6), as GGK-P decreases over time in the phosphorylation reaction when DNA is present. In summary, all three linker mutants are phosphorylated by EnvZ-P, but the effect of DNA on the GGK linker substitution is dramatic, causing an increase in turnover of GGK-P (24).…”

“…When EnvZ is the phosphodonor, the observed increase in OmpR-P formation upon the addition of DNA is due to a decrease in the phosphatase activity of EnvZ (14,24). We can quantify the effect of DNA on the phosphatase activity of EnvZ, using an assay that detects the release of P i (33).…”

“…Phosphorylation Assays and Measurement of ATPase Activity-Phosphorylation reactions were carried out with EnvZ115 as described (24). ATPase reactions were carried out as described (24).…”

ADP reduces the oxygen-binding affinity of a sensory histidine kinase, FixL: The possibility of an enhanced reciprocating kinase reaction

“…In the case of homodimeric FixL, the in situ ADP generated by the phosphorylation reaction appears to be responsible for the allosteric effect. It is also evident that, to ensure a turnover of phosphorylation reactions, the generated ADP should be simultaneously liberated from FixL when phosphoFixJ is dissociated to bind to the fixK and nifA promoters, similar to CheY (30) and OmpR (31).…”