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Predominantly buried residues in the response regulator Spo0F influence specific sensor kinase recognition.

ABSTRACT: Several alanine mutations in the response regulator Spo0F induce hypersporulation in Bacillus subtilis. L66A, I90A and H101A mutants are purported to be involved in contacts stabilizing the orientation of the alpha4-helix and hence the beta4-alpha4 kinase recognition loop. Y13A is thought to affect the orientation of the alpha1-helix and consequently phosphatase action. Using comparative NMR chemical shift analyses for these mutants, we have confirmed these suppositions and isolated residues in Spo0F critical in sensor kinases discrimination. In addition, we discuss how buried residues and intra-protein communication networks contribute to precise molecular recognition by ensuring that the correct surface is presented.

SUBMITTER: McLaughlin PD

PROVIDER: S-EPMC1987376 | biostudies-literature | 2007 Apr

REPOSITORIES: biostudies-literature

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Predominantly buried residues in the response regulator Spo0F influence specific sensor kinase recognition.

McLaughlin Patrick D PD Bobay Benjamin G BG Regel Erin J EJ Thompson Richele J RJ Hoch James A JA Cavanagh John J

FEBS letters 20070305 7

Several alanine mutations in the response regulator Spo0F induce hypersporulation in Bacillus subtilis. L66A, I90A and H101A mutants are purported to be involved in contacts stabilizing the orientation of the alpha4-helix and hence the beta4-alpha4 kinase recognition loop. Y13A is thought to affect the orientation of the alpha1-helix and consequently phosphatase action. Using comparative NMR chemical shift analyses for these mutants, we have confirmed these suppositions and isolated residues in ...[more]

PMID: 17350627

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Project description:BackgroundThe CpxA sensor kinase-CpxR response regulator two-component regulatory system is a sentinel of bacterial envelope integrity. Integrating diverse signals, it can alter the expression of a wide array of components that serve to shield the envelope from damage and to promote bacterial survival. In bacterial pathogens such as Yersinia pseudotuberculosis, this also extends to pathogenesis. CpxR is thought to dimerize upon phosphorylation by the sensor kinase CpxA. This phosphorylation enables CpxR binding to specific DNA sequences where it acts on gene transcription. As Cpx pathway activation is dependent on protein-protein interactions, we performed an interaction analysis of CpxR and CpxA from Y. pseudotuberculosis.ResultsCpxR full-length and truncated versions that either contained or lacked a putative internal linker were all assessed for their ability to homodimerize and interact with CpxA. Using an adenylate cyclase-based bacterial two hybrid approach, full-length CpxR readily engaged with CpxA. The CpxR N-terminus could also homodimerize with itself and with a full-length CpxR. A second homodimerization assay based upon the ?cI repressor also demonstrated that the CpxR C-terminus could homodimerize. While the linker was not specifically required, it enhanced CpxR homodimerization. Mutagenesis of cpxR identified the aspartate at residue 51, putative N-terminal coiled-coil and C-terminal winged-helix-turn-helix domains as mediators of CpxR homodimerization. Scrutiny of CpxA full-length and truncated versions revealed that dimerization involved the N-terminus and an internal dimerization and histidine phosphotransfer domain.ConclusionsThis interaction analysis mapped regions of CpxR and CpxA that were responsible for interactions with self or with each other. When combined with other physiological and biochemical tests both hybrid-based assays can be useful in dissecting molecular contacts that may underpin Cpx pathway activation and repression.

Project description:The facultative intracellular bacterial pathogen Francisella tularensis is the causative agent of tularemia in humans and animals. Gram-negative bacteria utilize two-component regulatory systems (TCS) to sense and respond to their changing environment. No classical, tandemly arranged sensor kinase and response regulator TCS genes exist in the human virulent Francisella tularensis subsp. tularensis, but orphaned members are present. PmrA is an orphan response regulator responsible for intramacrophage growth and virulence; however, the regulation of PmrA activity is not understood. We and others have shown that PmrA represses the expression of priM, described to encode an antivirulence determinant. By screening a mutant library for increased priM promoter activity, we identified the sensor kinase homolog QseC as an upstream regulator of priM expression, and this regulation is in part dependent upon the aspartate phosphorylation site of PmrA (D51). Several examined environmental signals, including epinephrine, which is reported to activate QseC in other bacteria, do not affect priM expression in a manner dependent on PmrA. Intramacrophage survival assays also question the finding that PriM is an antivirulence factor. Thus, these data suggest that the PmrA-regulated gene priM is modulated by the QseC-PmrA (QseB) TCS in Francisella IMPORTANCE The disease tularemia is caused by the highly infectious Gram-negative pathogen Francisella tularensis This bacterium encodes few regulatory factors (e.g., two-component systems [TCS]). PmrA, required for intramacrophage survival and virulence in the mouse model, is encoded by an orphan TCS response regulator gene. It is unclear how PmrA is responsive to environmental signals to regulate loci, including the PmrA-repressed gene priM We identify an orphan sensor kinase (QseC) that is required for priM repression and further explore both environmental signals that might regulate the QseC-PmrA TCS and the function of PriM.

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Predominantly buried residues in the response regulator Spo0F influence specific sensor kinase recognition.

Publications

Predominantly buried residues in the response regulator Spo0F influence specific sensor kinase recognition.

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