Comprehensive analysis of OmpR phosphorylation, dimerization, and DNA binding supports a canonical model for activation.

The OmpR/PhoB family of response regulators (RRs) is the largest class of two-component system signal transduction proteins. Extensive biochemical and structural characterization of these transcription factors has provided insights into their activation and DNA-binding mechanisms. For the most part, OmpR/PhoB family proteins are thought to become activated through phosphorylation from their cognate histidine kinase partners, which in turn facilitates an allosteric change in the RR, enabling homodimerization and subsequently enhanced DNA binding. Incongruently, it has been suggested that OmpR, the eponymous member of this RR family, becomes activated via different mechanisms, whereby DNA binding plays a central role in facilitating dimerization and phosphorylation. Characterization of the rate and extent of the phosphorylation of OmpR and OmpR DNA-binding mutants following activation of the EnvZ/OmpR two-component system shows that DNA binding is not essential for phosphorylation of OmpR in vivo. In addition, detailed analyses of the energetics of DNA binding and dimerization of OmpR in both its unphosphorylated and phosphorylated state indicate that phosphorylation enhances OmpR dimerization and that this dimerization enhancement is the energetic driving force for phosphorylation-mediated regulation of OmpR-DNA binding. These findings suggest that OmpR phosphorylation-mediated activation follows the same paradigm as the other members of the OmpR/PhoB family of RRs in contrast to previously proposed models of OmpR activation.