Signal transduction systems in bacteria provide the molecular basis for coupling environmental signals to appropriate adaptive responses. One of the most prevalent signaling strategies in bacteria is a phosphotransfer pathway between two-conserved proteins, a histidine protein kinase and a response regulator. These pathways, termed two-component systems, are widespread, with >9000 systems identified in -300 sequenced bacterial genomes to date. This project focuses on characterization of response regulators, proteins which function as phosphorylation-activated switches to control output responses of the systems. The OmpR/PhoB subfamily of response regulators, distinguished by a winged- helix DMA-binding domain, accounts for -one third of all response regulators and -half of all response regulator transcription factors. It has been recently established that OmpR/PhoB response regulators in their inactive states display different arrangements of their homologous domains, but upon phosphorylation adopt a common dimeric active state mediated by a conserved molecular surface. A primary aim of this project is to measure affinities for homo- and heterodimerization of OmpR/PhoB proteins using FRET to monitor interactions in vitro and in vivo to determine whether the common active state allows heterodimerization, providing a mechanism for integrating different two-component systems within a single cell.
A second aim i s to determine mechanisms through which different domain arrangements in inactive OmpR/PhoB proteins regulate their transition to an active state.
A third aim i s to characterize the complexity of transcriptional regulation by E. coli OmpR/PhoB response regulators on a genomic scale using a combination of structural, ChlP-on-chip, and bioinformatics analyses. Additional studies will focus on structural and functional characterization of protein-DNA interactions of OmpR/PhoB and LytTR response regulators. Relevance: In addition to their importance for basic competitiveness in natural environments, two- component signaling systems are often essential for virulence when pathogenic bacteria (e.g. Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enterica) infect their hosts. Hence, understanding the molecular details of signaling pathways and their protein components provides a foundation for the development of antimicrobial drugs.
|Gao, Rong; Stock, Ann M (2013) Evolutionary tuning of protein expression levels of a positively autoregulated two-component system. PLoS Genet 9:e1003927|
|Gao, Rong; Stock, Ann M (2013) Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling. Proc Natl Acad Sci U S A 110:672-7|
|Leonard, Paul G; Golemi-Kotra, Dasantila; Stock, Ann M (2013) Phosphorylation-dependent conformational changes and domain rearrangements in Staphylococcus aureus VraR activation. Proc Natl Acad Sci U S A 110:8525-30|
|Barbieri, Christopher M; Wu, Ti; Stock, Ann M (2013) Comprehensive analysis of OmpR phosphorylation, dimerization, and DNA binding supports a canonical model for activation. J Mol Biol 425:1612-26|
|Tang, Yat T; Gao, Rong; Havranek, James J et al. (2012) Inhibition of bacterial virulence: drug-like molecules targeting the Salmonella enterica PhoP response regulator. Chem Biol Drug Des 79:1007-17|
|Leonard, Paul G; Bezar, Ian F; Sidote, David J et al. (2012) Identification of a hydrophobic cleft in the LytTR domain of AgrA as a locus for small molecule interactions that inhibit DNA binding. Biochemistry 51:10035-43|
|Barbieri, Christopher M; Mack, Timothy R; Robinson, Victoria L et al. (2010) Regulation of response regulator autophosphorylation through interdomain contacts. J Biol Chem 285:32325-35|
|Gao, Rong; Stock, Ann M (2010) Molecular strategies for phosphorylation-mediated regulation of response regulator activity. Curr Opin Microbiol 13:160-7|
|Mack, Timothy R; Gao, Rong; Stock, Ann M (2009) Probing the roles of the two different dimers mediated by the receiver domain of the response regulator PhoB. J Mol Biol 389:349-64|
|Gao, Rong; Tao, Yuan; Stock, Ann M (2008) System-level mapping of Escherichia coli response regulator dimerization with FRET hybrids. Mol Microbiol 69:1358-72|
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