Comprehensive Analysis of OmpR Phosphorylation, Dimerization, and DNA Binding Supports a Canonical Model for Activation

Abstract

The OmpR/PhoB family of response regulators (RRs) is the largest class of two-component system (TCS) 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 (HK) 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 TCS 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.

Use of a Phosphorylation Site Mutant To Identify Distinct Modes of Gene Repression by the Control of Virulence Regulator (CovR) in Streptococcus pyogenes

“…The current model of OmpR/PhoB family regulator function predicates that response regulator dimerization is the driving force for phosphorylation-mediated gene regulation through binding to tandem DNAbinding sites (47)(48)(49). Our CovR-D53E transcription data suggest that CovR may employ additional regulatory mechanisms beyond that of typical OmpR/PhoB family members, the more so as in contrast to CovR many of them mainly function as transcriptional activators (50,51).…”

An Adaptive Mutation in Enterococcus faecium LiaR Associated with Antimicrobial Peptide Resistance Mimics Phosphorylation and Stabilizes LiaR in an Activated State

“…While members of the PhoB/OmpR family [29, 30] and FixJ [28, 31] utilize the hydrophobic surface of the α4-β5-α5 interface for phosphorylation triggered dimerization, the NarL transcription factor from E. coli [32], the cell-density-responsive transcription factor LuxR from S. aureus, S. aureus VraR [28] and B. subtilis DesR [27] employ a novel α1-α5 contact surface. An analogous involvement of helix α1 in intermolecular contacts has also been established in the M. tuberculosis NarL [33] and E.coli UhpA [34].…”

Trade-Offs in Delayed Information Transmission in Biochemical Networks

“…Conversely, the deactivation of x corresponds to unbinding of OmpR from the DNA, while the deactivation of z to the dephosphorylation of OmpR. Detailed experimental studies of the energetics of this system show that phosphorylation-activated dimerization drives an increase in DNA binding [66], suggesting that the biochemical regulation is a clockwise cycle such as presented in network E f of Fig. 8.…”