C-terminal DNA binding stimulates N-terminal phosphorylation of the outer membrane protein regulator OmpR from Escherichia coli

Abstract

Expression of the porin genes of Escherichia coli is regulated in part by the osmolarity of the growth medium. The process is controlled by the histidine kinase EnvZ and the response regulator OmpR. We have previously shown that phosphorylation of OmpR increases its affinity for the upstream regulatory regions of ompF and ompC. We now report that, in the presence of DNA, there is a dramatic stimulation in the level of phospho-OmpR. This effect is independent of the source of phosphorylation, i.e., stimulation of phosphorylation is observed with a small phosphorylating agent such as acetyl phosphate or with protein-catalyzed phosphorylation by the kinase EnvZ. The dephosphorylation rate of phospho-OmpR is affected only slightly by the presence of DNA; thus, the increased level is largely caused by an increased rate of phosphorylation. Stimulation of phosphorylation requires specific binding of DNA by OmpR. Occupancy of the DNA binding domain exposes a trypsin cleavage site in the linker, which connects the phosphorylation domain with the DNA binding domain. Our results indicate that when DNA binds in the C terminus, it enhances phosphorylation in the N terminus, and the linker undergoes a conformational change. A generalized mechanism involving a four-state model for response regulators is proposed.winged helix-turn-helix ͉ response regulator ͉ porin regulation ͉ two-component regulatory system ͉ transcriptional activator A ll organisms must communicate with their environment to survive. Two-component regulatory systems have emerged as a paradigm for adaptive responses. In its simplest form, a two-component system contains a sensor, a histidine kinase, and a response regulator, often a transcriptional activator. Changes in the environment result in phosphorylation of the sensor followed by transphosphorylation onto the response regulator. Adaptive responses controlled by two-component regulatory systems are diverse and include chemotaxis, fruit ripening, sporulation, and virulence gene expression in numerous pathogens (see ref. 1).The outer membrane proteins OmpF and OmpC in Escherichia coli are regulated in response to changes in the osmolarity of the medium (see ref. 2 for a recent review). At low osmolarity, OmpF predominates; at high osmolarity, ompF is repressed, and OmpC is the major porin in the outer membrane (3). This process is controlled by the EnvZ͞OmpR two-component regulatory system (4). EnvZ is a histidine kinase located in the inner membrane, OmpR is a cytoplasmic DNA binding protein (5). The two proteins communicate via a series of phosphorylation and phosphotransfer reactions. EnvZ senses the osmotic environment and is autophosphorylated from intracellular ATP at His-243 (6, 7). EnvZ-P phosphorylates OmpR at , and phospho-OmpR (OmpR-P) binds to the upstream sites of the porin genes to regulate their expression (9-12). EnvZ also stimulates the dephosphorylation of OmpR-P, thereby controlling the concentration of cellular OmpR-P (11, 13). Phosphorylation of OmpR results in an increase in its affin...

The critical role of DNA in the equilibrium between OmpR and phosphorylated OmpR mediated by EnvZ in Escherichiacoli

“…The above results indicate that OmpR-P binding to DNA is essential for the observed accumulation of OmpR-P by DNA, consistent with the previous observation (24). We further tested this notion with OmpR N (residues 1-134).…”

“…g OmpR was phosphorylated with 50 M [␥-32 P]ATP by 60 g glutathione S-transferase (GST)-EnvZc11, a GST fusion of a superkinase mutant, EnvZcT247R, in 200 l of buffer A containing 5 mM Ca 2ϩ at room temperature for 1 h. HPLC C4 column analysis (24) demonstrated that more than 95% OmpR in the reaction has been converted into OmpR-P (data not shown). The reaction mixture was stopped by addition of 10 mM EDTA and then applied to a Sephacryl S-100 HR gel-filtration column (0.8 ϫ 10 cm; Amersham Pharmacia) to separate GST-EnvZc11 from OmpR-P and to remove free [␥-32 P]ATP and inorganic phosphate.…”

“…Recently it was reported that phosphorylation of OmpR by either acetyl phosphate or EnvZ was enhanced in the presence of a DNA fragment containing OmpR-P-binding regions (24). On the basis of acetyl phosphate being only a phosphor donor for OmpR but not a phosphatase for OmpR-P and that the half-life of OmpR-P in the presence of DNA is slightly longer than that in the absence of DNA, it was concluded that DNA enhances the rate of phosphorylation of OmpR but not the rate of dephosphorylation of OmpR-P.…”

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

“…Because a linker of 20 amino acid residues should provide a similar increase in local concentration of the amino and carboxyl termini as a linker of 15 amino acid residues, it seems likely that the linker of OmpR plays some active role in directing the appropriate interaction between the two domains (13, 39, 40). Evidence supporting this view comes from our studies of limited proteolysis with trypsin, in which we found that cleavage sites in the linker were sensitive to both phosphorylation and DNA binding in the amino-and carboxylterminal domains, respectively (13,41).…”

“…Phosphorylation of OmpR at the aminoterminal aspartate 55 increases the DNA binding affinity of the carboxyl terminus (9 -12). Conversely, the presence of specific DNA binding sites increases the steady-state amount of OmpR-P formed in vitro (13,14). Our laboratory is interested in determining the mechanism responsible for this interdomain communication in OmpR.…”